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Neurodegenerative diseases are characterized by progressive dysfunction and loss of specific sets of neurons. While extensive research has focused on elucidating the genetic and epigenetic factors and molecular mechanisms underlying these disorders, emerging evidence highlights the critical role of secretion in the pathogenesis, possibly even onset, and progression of neurodegenerative diseases, suggesting the occurrence of non-cell-autonomous mechanisms. Secretion is a fundamental process that regulates intercellular communication, supports cellular homeostasis, and orchestrates various physiological functions in the body. Defective secretion can impair the release of neurotransmitters and other signaling molecules, disrupting synaptic transmission and compromising neuronal survival. It can also contribute to the accumulation, misfolding, and aggregation of disease-associated proteins, leading to neurotoxicity and neuronal dysfunction. In this review, we discuss the implications of defective secretion in the context of Parkinson's disease, emphasizing its role in protein aggregation, synaptic dysfunction, extracellular vesicle secretion, and neuroinflammation. We propose a multiple-hit model whereby protein accumulation and secretory defects must be combined for the onset and progression of the disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca 2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1 +/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca 2+ -dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Marine animals display diverse vibrant colors, but the mechanisms underlying their specific coloration remain to be clarified. Blue coloration is known to be achieved through a bathochromic shift of the orange carotenoid astaxanthin (AXT) by the crustacean protein crustacyanin, but other examples have not yet been well investigated. Here, we identified an ependymin (EPD)-related water-soluble blue carotenoprotein responsible for the specific coloration of the marine blue sponge Haliclona sp. EPD was originally identified in the fish brain as a protein involved in memory consolidation and neuronal regeneration. The purified blue protein, designated as EPD-related blue carotenoprotein-1, was identified as a secreted glycoprotein. We show that it consists of a heterodimer that binds orange AXT and mytiloxanthin and exhibits a bathochromic shift. Our crystal structure analysis of the natively purified EPD-related blue carotenoprotein-1 revealed that these two carotenoids are specifically bound to the heterodimer interface, where the polyene chains are aligned in parallel to each other like in β-crustacyanin, although the two proteins are evolutionary and structurally unrelated. Furthermore, using reconstitution assays, we found that incomplete bathochromic shifts occurred when the protein bound to only AXT or mytiloxanthin. Taken together, we identified an EPD in a basal metazoan as a blue protein that decorates the sponge body by binding specific structurally unrelated carotenoids., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61β/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article. M. S is an incumbent of the Dr Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Secretion systems utilize ATPase activity to facilitate the translocation of proteins into and across membranes. In bacteria, the universally conserved SecA ATPase binds a large repertoire of preproteins and interacts with the SecYEG translocon. In contrast, the type 7b secretion system (T7bSS) of Staphylococcus aureus supports the secretion of a restricted subset of proteins. T7bSSs are found in several Firmicutes as gene clusters encoding secreted WXG100 proteins and FtsK/SpoIIIE-like ATPase. In S. aureus, this ATPase is called EssC and comprises two cytosolic forkhead-associated domains (FHA 1-2 ), two membrane-spanning segments (TM 1-2 ), and four cytosolic modules named DUF (domain of unknown function) and ATPases 1-3  (D1D2D3). However, a detailed understanding of the interactions of EssC in the T7bSS is not clear. Here, we tagged EssC and performed affinity chromatography of detergent-solubilized extracts of wild type and isogenic mutants of S. aureus. We found that EssC recruits EsaA, EssA, and EssB in a complex referred to as the ESS (ESAT-6 like secretion system) translocon, and secreted substrates were not required for translocon assembly. Furthermore, deletions of FHA 1  and DUF rendered EssC unstable, whereas FHA 2  was required for association with EssB. This interaction was independent of EsaA, but EsaA was required to recruit EssA to the EssC-EssB complex. Finally, we show that assembly of the ESS translocon was impaired upon mutation of D2 structural motifs. Together, our data indicate that the ESS translocon is maintained fully assembled at the plasma membrane and that D2 is fundamental in sustaining the integrity of this complex., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Loss-of-function mutations in progranulin (GRN) are a major genetic cause of frontotemporal dementia (FTD), possibly due to loss of progranulin's neurotrophic and anti-inflammatory effects. Progranulin promotes neuronal growth and protects against excitotoxicity and other forms of injury. It is unclear if these neurotrophic effects are mediated through cellular signaling or through promotion of lysosomal function. Progranulin is a secreted proprotein that may activate neurotrophic signaling through cell-surface receptors. However, progranulin is efficiently trafficked to lysosomes and is necessary for maintaining lysosomal function. To determine which of these mechanisms mediates progranulin's protection against excitotoxicity, we generated lentiviral vectors expressing progranulin (PGRN) or lysosome-targeted progranulin (L-PGRN). L-PGRN was generated by fusing the LAMP-1 transmembrane and cytosolic domains to the C-terminus of progranulin. L-PGRN exhibited no detectable secretion, but was delivered to lysosomes and processed into granulins. PGRN and L-PGRN protected against NMDA excitotoxicity in rat primary cortical neurons, but L-PGRN had more consistent protective effects than PGRN. L-PGRN's protective effects were likely mediated through the autophagy-lysosomal pathway. In control neurons, an excitotoxic dose of NMDA stimulated autophagy, and inhibiting autophagy with 3-methyladenine reduced excitotoxic cell death. L-PGRN blunted the autophagic response to NMDA and occluded the protective effect of 3-methyladenine. This was not due to a general impairment of autophagy, as L-PGRN increased basal autophagy and did not alter autophagy after nutrient starvation. These data show that progranulin's protection against excitotoxicity does not require extracellular progranulin, but is mediated through lysosomes, providing a mechanistic link between progranulin's lysosomal and neurotrophic effects., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin's lymphoma (NHL) tissues of HIV-1-seropositive (HIV + ) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala-Ala) or 125 to 126 (Gly-Asn), respectively, are secreted from HIV-1-infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55 Gag ) by cellular aspartyl proteases. Binding of Pr55 Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55 Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out. However, the orthologous pectate lyases Pel3 and PelI from these bacteria, which share 67% of sequence identity, are not secreted by the counterpart T2SS of each bacterium, indicating a fine-tuned control of protein recruitment. To identify the related secretion determinants, we first performed a structural characterization and comparison of Pel3 with PelI using X-ray crystallography. Then, to assess the biological relevance of the observed structural variations, we conducted a loop-substitution analysis of Pel3 combined with secretion assays. We showed that there is not one element with a definite secondary structure but several distant and structurally flexible loop regions that are essential for the secretion of Pel3 and that these loop regions act together as a composite secretion signal. Interestingly, depending on the crystal contacts, one of these key secretion determinants undergoes disorder-to-order transitions that could reflect its transient structuration upon the contact with the appropriate T2SS components. We hypothesize that such T2SS-induced structuration of some intrinsically disordered zones of secretion substrates could be part of the recruitment mechanism used by T2SS., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5 mt -PPE4 mt -EspG 3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG 3mm interacts exclusively with PPE4 mt in a similar manner to EspG 5 , shielding the hydrophobic tip of PPE4 mt from solvent. The C-terminal helical domain of EspG 3mm is dynamic, alternating between "open" and "closed" forms, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE-PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE-PPE pairs., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Williamson et al.)

Viperin plays an important and multifaceted role in the innate immune response to viral infection. Viperin is also notable as one of very few radical SAM-dependent enzymes present in higher animals; however, the enzyme appears broadly conserved across all kingdoms of life, which suggests that it represents an ancient defense mechanism against viral infections. Although viperin was discovered some 20 years ago, only recently was the enzyme's structure determined and its catalytic activity elucidated. The enzyme converts CTP to 3'-deoxy-3',4'-didehydro-CTP, which functions as novel chain-terminating antiviral nucleotide when misincorporated by viral RNA-dependent RNA polymerases. Moreover, in higher animals, viperin interacts with numerous other host and viral proteins, and it is apparent that this complex network of interactions constitutes another important aspect of the protein's antiviral activity. An emerging theme is that viperin appears to facilitate ubiquitin-dependent proteasomal degradation of some of the proteins it interacts with. Viperin-targeted protein degradation contributes to the antiviral response either by down-regulating various metabolic pathways important for viral replication or by directly targeting viral proteins for degradation. Here, we review recent advances in our understanding of the structure and catalytic activity of viperin, together with studies investigating the interactions between viperin and its target proteins. These studies have provided detailed insights into the biochemical processes underpinning this unusual enzyme's wide-ranging antiviral activity. We also highlight recent intriguing reports that implicate a broader role for viperin in regulating nonpathological cellular processes, including thermogenesis and protein secretion., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ghosh and Marsh.)

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that "C-Pro-only" constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Doan et al.)

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella -containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication., (© 2020 Martinez et al.)

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli , the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo ., (© 2020 Cranford-Smith et al.)

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1-5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG 5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1., (© 2020 Damen et al.)

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding., (© 2020 Robinson et al.)

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins ( e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance., (© 2020 Jurick II et al.)

Porcupine O- acyltransferase (PORCN) is considered essential for Wnt secretion and signaling. However, we observed that PORCN inhibition does not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, here we overexpressed WNT4 or WNT3A in breast cancer, ovarian cancer, and fibrosarcoma cell lines. Conditioned media from these lines and co-culture systems were used to assess the dependence of Wnt secretion and activity on the critical Wnt secretion proteins PORCN and Wnt ligand secretion (WLS) mediator. We observed that WLS is universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across the cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not exhibit paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell-autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or Wnt secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated here may be critical in WNT4-driven cellular contexts or in those that are considered to have dysfunctional Wnt signaling., (© 2019 Rao et al.)

Pseudomonas aeruginosa secretes multiple proteases that are implicated in its pathogenesis, and most of them are regulated by quorum sensing (QS). In this study, we found that the activities of three major extracellular proteases, protease IV (PIV), elastase A (LasA), and elastase B (LasB), are reduced considerably when expressed in a QS mutant (MW1). PIV and LasA expressed in MW1 exhibited little activity, even when purified, and their activities were inhibited by noncleavage or binding of their propeptides. LasB was activated by a QS-dependent factor, indicating that, unlike what has been proposed previously, LasB is not autoactivated. When LasB was relieved from inhibition, it activated PIV, which then sequentially processed pro-LasA to mature LasA. When activated, LasB was not inhibited by exogenous addition of its propeptide, but LasA and PIV were inhibited by their propeptides, even after prior activation. These differences may be explained by the fact that LasB can degrade its own propeptide but PIV and LasA cannot. We also found that, although PIV is the preferred LasA-activating factor, LasB can also partially activate LasA. Overall, LasB, PIV, and LasA were activated postsecretionally in a cascading manner in which the initial activation of LasB was controlled tightly by QS at the protein level in addition to the well-known transcriptional control of these proteases by QS. Interestingly, human elastase also activated LasA, indicating that the activation cascade is triggered by host factors during infection. In summary, a QS-induced proteolytic cascade activates secreted proteases from P. aeruginosa., (© 2019 Li and Lee.)

The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli , here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG-interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD-SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely-overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is noncompetitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon., (© 2019 Jauss et al.)

Bacterial pathogens assemble adhesive surface structures termed pili or fimbriae to initiate and sustain infection of host tissues. Uropathogenic Escherichia coli , the primary causative agent of urinary tract infections, expresses type 1 and P pili required for colonization of the bladder and kidney, respectively. These pili are assembled by the conserved chaperone-usher (CU) pathway, in which a periplasmic chaperone works together with an outer membrane (OM) usher protein to build and secrete the pilus fiber. Previously, we found that the small molecule and antiparasitic drug nitazoxanide (NTZ) inhibits CU pathway-mediated pilus biogenesis in E. coli by specifically interfering with proper maturation of the usher protein in the OM. The usher is folded and inserted into the OM by the β-barrel assembly machine (BAM) complex, which in E. coli comprises five proteins, BamA-E. Here, we show that sensitivity of the usher to NTZ is modulated by BAM expression levels and requires the BamB and BamE lipoproteins. Furthermore, a genetic screen for NTZ-resistant bacterial mutants isolated a mutation in the essential BamD lipoprotein. These findings suggest that NTZ selectively interferes with an usher-specific arm of the BAM complex, revealing new details of the usher folding pathway and BAM complex function. Evaluation of a set of NTZ derivatives identified compounds with increased potency and disclosed that NTZ's nitrothiazole ring is critical for usher inhibition. In summary, our findings indicate highly specific effects of NTZ on the usher folding pathway and have uncovered NTZ analogs that specifically decrease usher levels in the OM., (© 2019 Psonis et al.)

Nonsynonymous gene mutations can be beneficial, neutral, or detrimental to the stability, structure, and biological function of the encoded protein, but the effects of these mutations are often not readily predictable. For example, the β-propeller olfactomedin domain of myocilin (mOLF) exhibits a complex interrelationship among structure(s), stability, and aggregation. Numerous mutations within mOLF are linked to glaucoma; the resulting variants are less stable, aggregation-prone, and sequestered intracellularly, causing cytotoxicity. Here, we report the first stable mOLF variants carrying substitutions in the calcium-binding site that exhibit solution characteristics indistinguishable from those of glaucoma variants. Crystal structures of these stable variants at 1.8-2.0-Å resolution revealed features that we could not predict by molecular dynamics simulations, including loss of loop structure, helix unwinding, and a blade shift. Double mutants that combined a stabilizing substitution and a selected glaucoma-causing single-point mutant rescued in vitro folding and stability defects. In the context of full-length myocilin, secretion of stable single variants was indistinguishable from that of the WT protein, and the double mutants were secreted to varying extents. In summary, our finding that mOLF can tolerate particular substitutions that render the protein stable despite a conformational switch emphasizes the complexities in differentiating between benign and glaucoma-causing variants and provides new insight into the possible biological function of myocilin., (© 2019 Hill et al.)

The E3 ubiquitin ligase membrane-associated ring-CH-type finger 2 (MARCH2) is known to be involved in intracellular vesicular trafficking, but its role in the early secretory pathway between the endoplasmic reticulum (ER) and Golgi compartments is largely unknown. Human ER-Golgi intermediate compartment protein 2 (ERGIC2) and ERGIC3 are orthologs of Erv41 and Erv46 in yeast, proteins that form a heteromeric complex, cycle between the ER and Golgi, and function as cargo receptors in both anterograde and retrograde protein trafficking. Here, we report that MARCH2 directs ubiquitination and subsequent degradation of ERGIC3 and that MARCH2 depletion increases endogenous ERGIC3 levels. We provide evidence that the lysine residues at positions 6 and 8 of ERGIC3 are the major sites of MARCH2-mediated ubiquitination. Of note, MARCH2 did not significantly decrease the levels of an ERGIC3 variant with lysine-to-arginine substitutions at residues 6 and 8. We also show that ERGIC3 binds to itself or to ERGIC2, whereas ERGIC2 is unable to interact with itself. Our results indicate that α 1 -antitrypsin and haptoglobin are likely to be cargo proteins of ERGIC3. We further observed that α 1 -antitrypsin and haptoglobin specifically bind to ERGIC3 and that ERGIC3 depletion decreases their secretion. Moreover, MARCH2 reduced secretion of α 1 -antitrypsin and haptoglobin, and coexpression of the ubiquitination-resistant ERGIC3 variant largely restored their secretion, suggesting that MARCH2-mediated ERGIC3 ubiquitination is the major cause of the decrease in trafficking of ERGIC3-binding secretory proteins. Our findings provide detailed insights into the regulation of the early secretory pathway by MARCH2 and into ERGIC3 function., (© 2019 Yoo et al.)

Biomineralization is a widespread biological process in the formation of shells, teeth, or bones. Matrix proteins in biominerals have been widely investigated for their roles in directing biomineralization processes such as crystal morphologies, polymorphs, and orientations. Here, we characterized a basic matrix protein, named mantle protein N25 (N25), identified previously in the Akoya pearl oyster ( Pinctada fucata ). Unlike some known acidic matrix proteins containing Asp or Glu as possible Ca 2+ -binding residues, we found that N25 is rich in Pro (12.4%), Ser (12.8%), and Lys (8.8%), suggesting it may perform a different function. We used the recombinant protein purified by refolding from inclusion bodies in a Ca(HCO 3 ) 2 supersaturation system and found that it specifically affects calcite morphologies. An X-ray powder diffraction (XRD) assay revealed that N25 could help delay the transformation of vaterites (a metastable calcium carbonate polymorph) to calcite. We also used fluorescence super-resolution imaging to map the distribution of N25 in CaCO 3 crystals and transfected a recombinant N25-EGFP vector into HEK-293T cells to mimic the native process in which N25 is secreted by mantle epithelial cells and integrated into mineral structures. Our observations suggest N25 specifically affects crystal morphologies and provide evidence that basic proteins lacking acidic groups can also direct biomineralization. We propose that the attachment of N25 to specific sites on CaCO 3 crystals may inhibit some crystal polymorphs or morphological transformation., (© 2019 Yang et al.)

α 1 -Antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at position 342 in the mature protein, resulting in the Z mutation of the AAT gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes, causing a toxic gain of function. ERdj3 is an ER luminal DnaJ homologue, which, along with calreticulin, directly interacts with misfolded ZAAT. We hypothesize that depletion of each of these chaperones will change the fate of ZAAT polymers. Our study demonstrates that calreticulin modulation reveals a novel ZAAT degradation mechanism mediated by exosomes. Using human PiZZ hepatocytes and K42, a mouse calreticulin-deficient fibroblast cell line, our results show ERdj3 and calreticulin directly interact with ZAAT in PiZZ hepatocytes. Silencing calreticulin induces calcium independent ZAAT-ERdj3 secretion through the exosome pathway. This co-secretion decreases ZAAT aggregates within the ER of hepatocytes. We demonstrate that calreticulin has an inhibitory effect on exosome-mediated ZAAT-ERdj3 secretion. This is a novel ZAAT degradation process that involves a DnaJ homologue chaperone bound to ZAAT. In this context, calreticulin modulation may eliminate the toxic gain of function associated with aggregation of ZAAT in lung and liver, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease., (© 2019 Khodayari et al.)

Mycobacteria use type VII secretion systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to ESX-5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, mycosin protease (MycP), is not part of the core complex but is essential for secretion, as it stabilizes this membrane complex. Here we investigated which MycP domains are required for this stabilization by producing hybrid constructs between MycP 1 and MycP 5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane domain are required for the ESX system-specific function of mycosins. In addition, we observed that the transmembrane domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP 5 -specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycP P1 , encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP 5 These results provide the first insight into the regions of mycosins involved in interaction with and stabilization of their respective ESX complexes., (© 2019 van Winden et al.)

The bacterial type III secretion system (T3SS) delivers virulence proteins, called effectors, into eukaryotic cells. T3SS comprises a transmembrane secretion apparatus and a complex network of specialized chaperones that target protein substrates to this secretion apparatus. However, the regulation of secretion switching from early (needle and inner rod) to middle (tip/filament and translocators) substrates is incompletely understood. Here, we investigated chaperone-mediated secretion switching from early to middle substrates in the T3SS encoded by Salmonella pathogenicity island 2 (SPI2), essential for systemic infection. Our findings revealed that the protein encoded by ssaH regulates the secretion of an inner rod and early substrate, SsaI. Structural modeling revealed that SsaH is structurally similar to class III chaperones, known to associate with proteins in various pathogenic bacteria. The SPI2 protein SsaE was identified as a class V chaperone homolog and partner of SsaH. A pulldown analysis disclosed that SsaH and SsaE form a heterodimer, which interacted with another early substrate, the needle protein SsaG. Moreover, SsaE also helped stabilize SsaH and a middle substrate, SseB. We also found that SsaE regulates cellular SsaH levels to translocate the early substrates SsaG and SsaI and then promotes the translocation of SseB by stabilizing it. In summary, our results indicate that the class III chaperone SsaH facilitates SsaI secretion, and a heterodimer of SsaH and the type V chaperone SsaE then switches secretion to SsaG. This is the first report of a chaperone system that regulates both early and middle substrates during substrate switching for T3SS assembly., (© 2019 Takaya et al.)

Legionella pneumophila causes Legionnaires' disease, a severe form of pneumonia. L. pneumophila translocates more than 300 effectors into host cells via its Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) type IV secretion system to enable its replication in target cells. Here, we studied the effector LtpM, which is encoded in a recombination hot spot in L. pneumophila Paris. We show that a C-terminal phosphoinositol 3-phosphate (PI3P)-binding domain, also found in otherwise unrelated effectors, targets LtpM to the Legionella -containing vacuole and to early and late endosomes. LtpM expression in yeast caused cytotoxicity. Sequence comparison and structural homology modeling of the N-terminal domain of LtpM uncovered a remote similarity to the glycosyltransferase (GT) toxin PaTox from the bacterium Photorhabdus asymbiotica ; however, instead of the canonical DxD motif of GT-A type glycosyltransferases, essential for enzyme activity and divalent cation coordination, we found that a DxN motif is present in LtpM. Using UDP-glucose as sugar donor, we show that purified LtpM nevertheless exhibits glucohydrolase and autoglucosylation activity in vitro and demonstrate that PI3P binding activates LtpM's glucosyltransferase activity toward protein substrates. Substitution of the aspartate or the asparagine in the DxN motif abolished the activity of LtpM. Moreover, whereas all glycosyltransferase toxins and effectors identified so far depend on the presence of divalent cations, LtpM is active in their absence. Proteins containing LtpM-like GT domains are encoded in the genomes of other L. pneumophila isolates and species, suggesting that LtpM is the first member of a novel family of glycosyltransferase effectors employed to subvert hosts., (© 2019 Levanova et al.)

In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpY L and XcpZ M , of the T2SS assembly platform (AP) in the opportunistic pathogen Pseudomonas aeruginosa Competition experiments indicated that CbpD binding to XcpY L is XcpZ M -dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein-protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZ M homodimerization and increases the affinity between XcpY L and XcpZ M The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Many proteins in organelles of the secretory pathway, as well as secretory proteins, are translocated across and inserted into the endoplasmic reticulum membrane by the Sec61 translocon, a protein-conducting channel. The channel consists of 10 transmembrane (TM) segments of the Sec61α subunit and possesses an opening between TM2b and TM7, termed the lateral gate. Structural and biochemical analyses of complexes of Sec61 and its ortholog SecY have revealed that the lateral gate is the exit for signal sequences and TM segments of translocating polypeptides to the lipid bilayer and also involved in the recognition of such hydrophobic sequences. Moreover, even marginally hydrophobic (mH) segments insufficient for membrane integration can be transiently stalled in surrounding Sec61α regions and cross-linked to them, but how the Sec61 translocon accommodates these mH segments remains unclear. Here, we used Cys-scanned variants of human Sec61α expressed in cultured 293-H cells to examine which channel regions associate with mH segments. A TM segment in a ribosome-associated polypeptide was mainly cross-linked to positions at the lateral gate, whereas an mH segment in a nascent chain was cross-linked to the Sec61α pore-interior positions at TM5 and TM10, as well as the lateral gate. Of note, cross-linking at position 180 in TM5 of Sec61α was reduced by an I179A substitution. We therefore conclude that at least two Sec61α regions, the lateral gate and the pore-interior site around TM5, interact with mH segments and are involved in accommodating them., (© 2018 Kida and Sakaguchi.)

Adhesive pili are external component of fibrous adhesive organelles and help bacteria attach to biotic or abiotic surfaces. The biogenesis of adhesive pili via the chaperone-usher pathway (CUP) is independent of external energy sources. In the classical CUP, chaperones transport assembly-competent pilins in a folded but expanded conformation. During donor-strand exchange, pilins subsequently collapse, producing a tightly packed hydrophobic core and releasing the necessary free energy to drive fiber formation. Here, we show that pilus biogenesis in non-classical, archaic, and alternative CUPs uses a different source of conformational energy. High-resolution structures of the archaic Csu-pili system from Acinetobacter baumannii revealed that non-classical chaperones employ a short donor strand motif that is insufficient to fully complement the pilin fold. This results in chaperone-bound pilins being trapped in a substantially unfolded intermediate. The exchange of this short motif with the longer donor strand from adjacent pilin provides the full steric information essential for folding, and thereby induces a large unfolded-to-folded conformational transition to drive assembly. Our findings may inform the development of anti-adhesion drugs (pilicides) to combat bacterial infections., (© 2018 Pakharukova et al.)

In eukaryotic cells, elimination of misfolded proteins is essential for maintaining protein homeostasis and cell viability. Misfolding-associated protein secretion (MAPS) is a protein quality-control mechanism that exports misfolded cytosolic proteins via a compartment characteristic of late endosomes, but how cytosolic proteins enter this compartment is unclear. Because chaperone-mediated autophagy (CMA) is a known mechanism that imports cytosolic proteins bearing a specific CMA motif to lysosomes for degradation and because late endosomes and lysosomes overlap significantly in mammalian cells, we determined here whether CMA is involved in targeting protein cargoes to the lumen of late endosomes in MAPS. Using HEK293T and COS-7 cells and immunoblotting and -staining and coimmunoprecipitation methods, we show that, unlike CMA, the secretion of misfolded proteins in MAPS does not require cargo unfolding, is inhibited by serum starvation, and is not dependent on the CMA motif in cargo. Intriguingly, knockdown of lysosome-associated membrane protein 2 (LAMP2), which consists of three isoforms, including a variant proposed to form a protein channel on lysosomes for CMA, attenuated MAPS. However, this could not be attributed to the proposed channel function of the LAMP2a isoform because overexpression of a cytosolic MAPS stimulator, DnaJ heat shock protein family (Hsp40) member C5 (DNAJC5), fully rescued the secretion defect associated with LAMP2 deficiency. We conclude that, in MAPS, cargoes use a CMA-independent mechanism to enter a nondegradative prelysosomal compartment., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Many bacterial pathogens employ multicomponent protein complexes such as type IV secretion systems (T4SSs) to transfer virulence factors into host cells. Here we studied the interaction between two essential T4SS components: the very hydrophobic inner membrane protein VirB6, which may be a component of the translocation channel, and VirB10, which links the inner and outer bacterial membranes. To map the interaction site between these two T4SS components, we conducted alanine scanning and deleted six-amino acid stretches from the N-terminal periplasmic domain of VirB6 from Brucella suis Using the bacterial two-hybrid system to analyze the effects of these alterations on the VirB6-VirB10 interaction, we identified the amino acid regions 16-21 and 28-33 and Leu-18 in VirB6 as being required for this interaction. SDS-PAGE coupled with Western blotting of cell lysates and native PAGE of detergent-extracted membrane proteins revealed that the corresponding VirB6 residues in Agrobacterium tumefaciens (Phe-20 and amino acids 18-23 and 30-35) modulate the stability of both VirB6 and VirB5. However, the results from immuno-EM and super-resolution microscopy suggested that these regions and residues are not required for membrane association or for polar localization of VirB6. The six-amino acid deletions in the N terminus of VirB6 abolished pilus formation and virulence of A. tumefaciens , and the corresponding deletions in the VirB6 homolog TraD from the plasmid pKM101-T4SS abrogated plasmid transfer. Our results indicate that specific residues of the VirB6 N-terminal domain are required for VirB6 stabilization, its interaction with VirB10, and the incorporation of VirB2 and VirB5 into T-pili., (© 2018 Mary et al.)

Human myeloid-derived growth factor (MYDGF; also known as C19orf10) is named based on its identification as a secreted monocyte/macrophage-derived mediator of cardiac repair following myocardial infarction in mice. Homologs of MYDGF, however, are present in organisms throughout and outside of the animal kingdom, some of which lack hematopoietic and circulatory systems. Moreover, the UPF0556 protein domain, which defines these homologs, lacks a known structure. As a result, the functions and properties of MYDGF are unclear. Our current work was initiated to test whether MYDGF is present in secretory vesicles of eosinophils as it was recently reported to be abundant in these cells. However, we could not demonstrate secretion and unexpectedly discovered that MYDGF colocalizes with P4HB in the nuclear envelope, which comprises the bulk of endoplasmic reticulum (ER) in eosinophils, and with P4HB and RCAS1 in Golgi. We noted a ubiquitous C-terminal sequence, BX EL ( B , basic; X , variable residue; E, Glu; L, Leu), that has the potential to retain human MYDGF and its homologs in the ER. To test the functionality of this sequence, we expressed full-length human MYDGF or MYDGF lacking the C-terminal Glu-Leu residues in monolayers of human embryonic kidney 293 (HEK293) cells. Full-length MYDGF accumulated in cells, whereas truncated MYDGF appeared in the medium. These observations reveal that MYDGF resides in the ER and Golgi and provide a new framework for investigating and understanding this intriguing protein., (© 2018 Bortnov et al.)

Major histocompatibility complex-I-β 2 m dimers (MHC-I) bind peptides derived from intracellular proteins, enabling the immune system to distinguish between normal cells and those expressing pathogen-derived or mutant proteins. The peptides bind to MHC-I in the endoplasmic reticulum (ER), and this binding is facilitated by the p eptide l oading c omplex (PLC), which contains calreticulin (CRT). CRT associates with MHC-I via a conserved glycan present on MHC-I and recruits it to the PLC for peptide binding. Somatic frameshift mutations in CRT (CRT-FS) drive the proliferation of a subset of myeloproliferative neoplasms, which are chronic blood tumors. All CRT-FS proteins have a C-terminal sequence lacking the normal ER-retention signal and possessing a net negative charge rather than the normal positive charge. We characterized the effect of CRT-FS on antigen presentation by MHC-I in human cells. Our results indicate that CRT-FS cannot mediate CRT's peptide loading function in the PLC. Cells lacking CRT exhibited reduced surface MHC-I levels, consistent with reduced binding of high-affinity peptides, and this was not reversed by CRT-FS expression. CRT-FS was secreted and not detectably associated with the PLC, leading to poor MHC-I recruitment, although CRT-FS could still associate with MHC-I in a glycan-dependent manner. The addition of an ER-retention sequence to CRT-FS restored its association with the PLC but did not rescue MHC-I recruitment or its surface expression, indicating that the CRT-FS mutants functionally compromise the PLC. MHC-I down-regulation permits tumor cells to evade immune surveillance, and these findings may therefore be relevant for designing effective immunotherapies for managing myeloproliferative neoplasms., (© 2018 Arshad and Cresswell.)

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens , T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa , PppA and TagF are two distinct proteins that repress T6SS in TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens , but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans -Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations., (© 2018 Ma et al.)

Many pathogenic bacteria, including Streptococcus gordonii , possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O -glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1-3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O -glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N -acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery.

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates ( N -glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N -glycans (23%). These proportions indicated restricted N -glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N -glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N -glycoforms similar to NK cell-derived CD16a but yielded N -glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N -glycan composition affected antibody binding: CD16a with oligomannose N -glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N -glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N -glycan composition and antibody-binding affinity., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) down-regulates the low-density lipoprotein (LDL) receptor, elevating LDL cholesterol and accelerating atherosclerotic heart disease, making it a promising cardiovascular drug target. To achieve its maximal effect on the LDL receptor, PCSK9 requires autoproteolysis. After cleavage, PCSK9 retains its prodomain in the active site as a self-inhibitor. Unlike other proprotein convertases, however, this retention is permanent, inhibiting any further protease activity for the remainder of its life cycle. Such inhibition has proven a major challenge toward a complete biochemical characterization of PCSK9's proteolytic function, which could inform therapeutic approaches against its hypercholesterolemic effects. To address this challenge, we employed a cell-based, high-throughput method using a luciferase readout to evaluate the single-turnover PCSK9 proteolytic event. We combined this method with saturation mutagenesis libraries to interrogate the sequence specificities of PCSK9 cleavage and proteolysis-independent secretion. Our results highlight several key differences in sequence identity between these two steps, complement known structural data, and suggest that PCSK9 self-proteolysis is the rate-limiting step of secretion. Additionally, we found that for missense SNPs within PCSK9, alterations in both proteolysis and secretion are common. Last, we show that some SNPs allosterically modulate PCSK9's substrate sequence specificity. Our findings indicate that PCSK9 proteolysis acts as a commonly perturbed but critical switch in controlling lipid homeostasis and provide a new hope for the development of small-molecule PCSK9 inhibitors., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The bacterial type VI secretion system (T6SS) mediates antagonistic cell-cell interactions between competing Gram-negative bacteria. In plant-beneficial bacteria, this pathway has been shown to suppress the growth of bacterial pathogens; however, the identification and mode of action of T6SS effector proteins that mediate this protective effect remain poorly defined. Here, we identify two previously uncharacterized effectors required for interbacterial antagonism by the plant commensal bacterium Pseudomonas protegens Consistent with the established effector-immunity paradigm for antibacterial T6SS substrates, the toxic activities of these effectors are neutralized by adjacently encoded cognate immunity determinants. Although one of these effectors, RhsA, belongs to the family of DNase enzymes, the activity of the other was not apparent from its sequence. To determine the mechanism of toxicity of this latter effector, we determined its 1.3 Å crystal structure in complex with its immunity protein and found that it resembles NAD(P) + -degrading enzymes. In line with this structural similarity, biochemical characterization of this effector, termed Tne2 ( T ype VI secretion N ADase e ffector family 2), demonstrates that it possesses potent NAD(P) + hydrolase activity. Tne2 is the founding member of a widespread family of interbacterial NADases predicted to transit not only the Gram-negative T6SS but also the Gram-positive type VII secretion system, a pathway recently implicated in interbacterial competition among Firmicutes. Together, this work identifies new T6SS effectors employed by a plant commensal bacterium to antagonize its competitors and broadly implicates NAD(P) + -hydrolyzing enzymes as substrates of interbacterial conflict pathways found in diverse bacterial phyla., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The type VI secretion system (T6SS) delivers toxic effectors between Gram-negative bacteria. Most antibacterial T6SS effectors are peptidoglycanases, nucleases, or lipases. In the current work, Tang et al. structurally and functionally characterize a novel family of NAD(P) + -hydrolyzing effectors (NADases), thus expanding the documented types of T6SS substrates. Bioinformatic identification of NADase family members putatively secreted by the bacteriolytic type VII secretion system (T7SS) of Gram-positive bacteria further points to NADases as a diverse and important class of effectors., Competing Interests: The authors declare that they have no conflicts of interests with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health., (© 2018 Lopez and Feldman.)

N -Glycosylation plays a fundamental role in many biological processes. Human diamine oxidase (hDAO), required for histamine catabolism, has multiple N- glycosylation sites, but their roles, for example in DAO secretion, are unclear. We recently reported that the N- glycosylation sites Asn-168, Asn-538, and Asn-745 in recombinant hDAO (rhDAO) carry complex-type glycans, whereas Asn-110 carries only mammalian-atypical oligomannosidic glycans. Here, we show that Asn-110 in native hDAO from amniotic fluid and Caco-2 cells, DAO from porcine kidneys, and rhDAO produced in two different HEK293 cell lines is also consistently occupied by oligomannosidic glycans. Glycans at Asn-168 were predominantly sialylated with bi- to tetra-antennary branches, and Asn-538 and Asn-745 had similar complex-type glycans with some tissue- and cell line-specific variations. The related copper-containing amine oxidase human vascular adhesion protein-1 also exclusively displayed high-mannose glycosylation at Asn-137. X-ray structures revealed that the residues adjacent to Asn-110 and Asn-137 form a highly conserved hydrophobic cleft interacting with the core trisaccharide. Asn-110 replacement with Gln completely abrogated rhDAO secretion and caused retention in the endoplasmic reticulum. Mutations of Asn-168, Asn-538, and Asn-745 reduced rhDAO secretion by 13, 71, and 32%, respectively. Asn-538/745 double and Asn-168/538/745 triple substitutions reduced rhDAO secretion by 85 and 94%. Because of their locations in the DAO structure, Asn-538 and Asn-745 glycosylations might be important for efficient DAO dimer formation. These functional results are reflected in the high evolutionary conservation of all four glycosylation sites. Human DAO is abundant only in the gastrointestinal tract, kidney, and placenta, and glycosylation seems essential for reaching high enzyme expression levels in these tissues., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

About one-quarter to nearly one-third of the proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Translocation of secretory proteins into the lumen of the endoplasmic reticulum or the periplasm of bacteria is mediated by a highly conserved heterotrimeric membrane protein complex denoted Sec61 in eukaryotes and SecYEG in bacteria. To evaluate a possible modulation of the translocation efficiency by secondary structures of the nascent peptide chain, we performed a comparative analysis in bacteria, yeast, and mammalian cells. Strikingly, neither the bacterial SecY nor the eukaryotic Sec61 translocon was able to efficiently transport proteins entirely composed of intrinsically disordered domains (IDDs) or β-strands. However, translocation could be restored by α-helical domains in a position- and organism-dependent manner. In bacteria, we found that the α-helical domains have to precede the IDD or β-strands, whereas in mammalian cells, C-terminally located α-helical domains are sufficient to promote translocation. Our study reveals an evolutionarily conserved deficiency of the Sec61/SecY complex to translocate IDDs and β-strands in the absence of α-helical domains. Moreover, our results may suggest that adaptive pathways co-evolved with the expansion of IDDs in the proteome of eukaryotic cells to increase the transport capacity of the Sec61 translocon., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Fusobacterium nucleatum is an oral pathogen that is linked to multiple human infections and colorectal cancer. Strikingly, F. nucleatum achieves virulence in the absence of large, multiprotein secretion systems (Types I, II, III, IV, and VI), which are widely used by Gram-negative bacteria for pathogenesis. By contrast, F. nucleatum strains contain genomic expansions of Type V secreted effectors (autotransporters) that are critical for host cell adherence, invasion, and biofilm formation. Here, we present the first characterization of an F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). Biochemical analysis of multiple Fusobacterium strains revealed that FplA is expressed as a full-length 85-kDa outer membrane-embedded protein or as a truncated phospholipase domain that remains associated with the outer membrane. Whereas the role of Type Vd secretion in bacteria remains unidentified, we show that FplA binds with high affinity to host phosphoinositide-signaling lipids, revealing a potential role for this enzyme in establishing an F. nucleatum intracellular niche. To further analyze the role of FplA, we developed an fplA gene knock-out strain, which will guide future in vivo studies to determine its potential role in F. nucleatum pathogenesis. In summary, using recombinant FplA constructs, we have identified a biochemical toolbox that includes lipid substrates for enzymatic assays, potent inhibitors, and chemical probes to detect, track, and characterize the role of Type Vd secreted phospholipases in Gram-negative bacteria., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis, has developed multiple strategies to adapt to the human host. The five type VII secretion systems, ESX-1-5, direct the export of many virulence-promoting protein effectors across the complex mycobacterial cell wall. One class of ESX substrates is the PE-PPE family of proteins, which is unique to mycobacteria and essential for infection, antigenic variation, and host-pathogen interactions. The genome of Mtb encodes 168 PE-PPE proteins. Many of them are thought to be secreted through ESX-5 secretion system and to function in pairs. However, understanding of the specific pairing of PE-PPE proteins and their structure-function relationship is limited by the challenging purification of many PE-PPE proteins, and our knowledge of the PE-PPE interactions therefore has been restricted to the PE25-PPE41 pair and its complex with the ESX-5 secretion system chaperone EspG5. Here, we report the crystal structure of a new PE-PPE pair, PE8-PPE15, in complex with EspG5. Our structure revealed that the EspG5-binding sites on PPE15 are relatively conserved among Mtb PPE proteins, suggesting that EspG5-PPE15 represents a more typical model for EspG5-PPE interactions than EspG5-PPE41. A structural comparison with the PE25-PPE41 complex disclosed conformational changes in the four-helix bundle structure and a unique binding mode in the PE8-PPE15 pair. Moreover, homology-modeling and mutagenesis studies further delineated the molecular determinants of the specific PE-PPE interactions. These findings help develop an atomic algorithm of ESX-5 substrate recognition and PE-PPE pairing., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)