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Two series of macrocyclic inhibitors addressing the S1 pocket and the prime site of the fibrinolytic serine protease plasmin have been developed. In the first series the P1 tranexamoyl residue was coupled to 4-aminophenylalanine in P1' position, which provided moderately potent inhibitors with inhibition constants around 1 µM. In the second series, a substituted biphenylalanine was incorporated as P1' residue leading to approximately 1000-fold stronger plasmin inhibitors, the best compounds possess subnanomolar inhibition constants. The most effective compounds already exhibit a certain selectivity as plasmin inhibitors compared to other trypsin-like serine proteases such as trypsin, plasma kallikrein, thrombin, activated protein Ca, as well as factors XIa and Xa. For inhibitor 28 of the second series, the co-crystal structure in complex with a Ser195Ala microplasmin mutant revealed the P2' residue adopts multiple conformations. Most polar contacts to plasmin and surrounding water molecules are mediated through the P1 tranexamoyl residue, whereas the bound conformation of the macrocycle is mainly stabilized by two intramolecular hydrogen bonds., (© 2024 Wiley‐VCH GmbH.)

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19 F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands., Competing Interests: Declaration of competing 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 © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant "closed" conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an "open" form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the K open of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg-R K . Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.open of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg-R KT . Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.

This paper introduces OpenFIBSEM, a universal API to control Focused Ion Beam Scanning Electron Microscopes (FIBSEM). OpenFIBSEM aims to improve the programmability and automation of electron microscopy workflows in structural biology research. The API is designed to be cross-platform, composable, and extendable: allowing users to use any portion of OpenFIBSEM to develop or integrate with other software tools. The package provides core functionality such as imaging, movement, milling, and manipulator control, as well as system calibration, alignment, and image analysis modules. Further, a library of reusable user interface components integrated with napari is provided, ensuring easy and efficient application development. OpenFIBSEM currently supports ThermoFisher and TESCAN hardware, with support for other manufacturers planned. To demonstrate the improved automation capabilities enabled by OpenFIBSEM, several example applications that are compatible with multiple hardware manufacturers are discussed. We argue that OpenFIBSEM provides the foundation for a cross-platform operating system and development ecosystem for FIBSEM systems. The API and applications are open-source and available on GitHub (https://github.com/DeMarcoLab/fibsem)., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alex de Marco reports financial support was provided by Australian Research Data Commons (XN006). James Whisstock reports financial support was provided by Australian Research Council (grant ID FL180100019)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Conjugative DNA transfer is a major factor in the dissemination of antibiotic resistance and virulence genes. In the Gram-positive pathogen Clostridium perfringens, the majority of conjugative plasmids share the conserved tcp locus that governs the assembly of the transfer system. Here, we describe multiple structures of the coupling protein TcpA, an essential ATPase that is suggested to provide the mechanical force to propel the DNA through the transfer apparatus. The structures of TcpA in the presence and absence of nucleotides revealed conformational rearrangements and highlight a crucial role for the unstructured C terminus. Our findings reveal that TcpA shares most structural similarity with the FtsK DNA translocase, a central component of the bacterial cell division machinery. Our structural data suggest that conjugation in C. perfringens may have evolved from the bacterial chromosome segregation system and, accordingly, suggest the possibility that double-stranded DNA is transferred through the Tcp conjugation apparatus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Two series of macrocyclic plasmin inhibitors with a C-terminal benzylamine group were synthesized. The substitution of the N-terminal phenylsulfonyl group of a previously described inhibitor provided two analogues with sub-nanomolar inhibition constants. Both compounds possess a high selectivity against all other tested trypsin-like serine proteases. Furthermore, a new approach was used to selectively introduce asymmetric linker segments. Two of these compounds inhibit plasmin with K i values close to 2 nM. For the first time, four crystal structures of these macrocyclic inhibitors could be determined in complex with a Ser195Ala microplasmin mutant. The macrocyclic core segment of the inhibitors binds to the open active site of plasmin without any steric hindrance. This binding mode is incompatible with other trypsin-like serine proteases containing a sterically demanding 99-hairpin loop. The crystal structures obtained experimentally explain the excellent selectivity of this inhibitor type as previously hypothesized., (© 2023 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11., (© 2023. The Author(s).)

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin β, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin β reveal unique features of the active site of meprin α, and helical assembly more broadly., (© 2022. The Author(s).)

To control infections phagocytes can directly kill invading microbes. Macrophage-expressed gene 1 (Mpeg1), a pore-forming protein sometimes known as perforin-2, is reported to be essential for bacterial killing following phagocytosis. Mice homozygous for the mutant allele Mpeg1 tm1Pod succumb to bacterial infection and exhibit deficiencies in bacterial killing in vitro. Here we describe a new Mpeg mutant allele Mpeg1 tm1.1Pib on the C57BL/6J background. Mice homozygous for the new allele are not abnormally susceptible to bacterial or viral infection, and irrespective of genetic background show no perturbation in bacterial killing in vitro. Potential reasons for these conflicting findings are discussed. In further work, we show that cytokine responses to inflammatory mediators, as well as antibody generation, are also normal in Mpeg1 tm1.1Pib/tm1.1Pib mice. We also show that Mpeg1 is localized to a CD68-positive endolysosomal compartment, and that it exists predominantly as a processed, two-chain disulfide-linked molecule. It is abundant in conventional dendritic cells 1, and mice lacking Mpeg1 do not present the model antigen ovalbumin efficiently. We conclude that Mpeg1 is not essential for innate antibacterial protection or antiviral immunity, but may play a focused role early in the adaptive immune response., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

P-Rex (PI(3,4,5)P 3 -dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gβγ and PI(3,4,5)P 3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gβγ and PI(3,4,5)P 3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling., (© 2022. Crown.)

Macrophages are an ancient blood cell lineage critical for homeostasis and defence against pathogens. Although their numbers were long thought to be sustained solely by haematopoietic organs, it has recently become clear that their proliferation, or self-renewal, also plays a major role. In the Drosophila larva, macrophages undergo a phase of rapid self-renewal, making this an attractive model for elucidating the signals and regulatory mechanisms involved. However, a central self-renewal pathway has not been identified in this system. Here, we show that the PDGF- and VEGF-receptor related (Pvr) pathway fulfils this role. Our data show that two of the three known Pvr ligands, PDGF- and VEGF-related factor 2 (Pvf2) and Pvf3, are major determinants of overall macrophage numbers, yet they each act in a temporally independent manner and via distinct mechanisms. While Pvf3 is needed prior to the self-renewal period, we find that Pvf2 is critical specifically for expanding the larval macrophage population. We further show that Pvf2 is a potent macrophage mitogen that is kept at limiting quantities by its transient expression in a remarkably small number of blood cells. Together, these data support a novel mechanism for the regulation of macrophage self-renewal rates by the dynamic transcriptional control of Pvf2. Given the strong parallels that exist between Drosophila and vertebrate macrophage systems, it is likely that a similar self-renewal control mechanism is at play across animal phyla., (© 2022 Federation of European Biochemical Societies.)

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates., (© 2022 by The American Society of Hematology.)

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics., Competing Interests: Declaration of interests Monash University, Hudson Institute (J.C.W., M.F.N., C.A.N.-P., and A.M.E.), and F. Hoffmann-La Roche (A.B., F.S., G.T., and L.L.) hold two patent families on IL-37, namely PCT/AU2016/050495 (Monash and Hudson only) and PCT/EP2020/087031 (Monash, Hudson, and Roche). There are no other conflicts of interest for these authors. All other authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Protein structure fundamentally underpins the function and processes of numerous biological systems. Fold recognition algorithms offer a sensitive and robust tool to detect structural, and thereby functional, similarities between distantly related homologs. In the era of accurate structure prediction owing to advances in machine learning techniques and a wealth of experimentally determined structures, previously curated sequence databases have become a rich source of biological information. Here, we use bioinformatic fold recognition algorithms to scan the entire AlphaFold structure database to identify novel protein family members, infer function and group predicted protein structures. As an example of the utility of this approach, we identify novel, previously unknown members of various pore-forming protein families, including MACPFs, GSDMs and aerolysin-like proteins., Competing Interests: The authors have declared that no competing interests exist.

Neurofibromin (NF1) mutations cause neurofibromatosis type 1 and drive numerous cancers, including breast and brain tumors. NF1 inhibits cellular proliferation through its guanosine triphosphatase-activating protein (GAP) activity against rat sarcoma (RAS). In the present study, cryo-electron microscope studies reveal that the human ~640-kDa NF1 homodimer features a gigantic 30 × 10 nm array of α-helices that form a core lemniscate-shaped scaffold. Three-dimensional variability analysis captured the catalytic GAP-related domain and lipid-binding SEC-PH domains positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long-sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Collectively these findings present potential new routes for therapeutic modulation of the RAS pathway., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P 3 to PI(4,5)P 2 to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by Gβγ subunits downstream of G protein-coupled receptor signaling and by PI(3,4,5)P 3 downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation. Here, using cross-linking mass spectrometry and functional studies, we gained mechanistic insights into PTEN:P-Rex2 complex assembly and coinhibition. We found that PTEN was anchored to P-Rex2 by interactions between the PDZ-interacting motif in the PTEN C-terminal tail and the second PDZ domain of P-Rex2. This interaction bridged PTEN across the P-Rex2 surface, preventing PI(3,4,5)P 3 hydrolysis. Conversely, PTEN both allosterically promoted an autoinhibited conformation of P-Rex2 and blocked its binding to Gβγ. In addition, we observed that the PTEN-deactivating mutations and P-Rex2 truncations combined to drive Rac1 activation to a greater extent than did either single variant alone. These insights enabled us to propose a class of gain-of-function, cancer-associated mutations within the PTEN:P-Rex2 interface that uncouple PTEN from the inhibition of Rac1 signaling., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Recent research in predicting protein secondary structure populations (SSP) based on Nuclear Magnetic Resonance (NMR) chemical shifts has helped quantitatively characterise the structural conformational properties of intrinsically disordered proteins and regions (IDP/IDR). Different from protein secondary structure (SS) prediction, the SSP prediction assumes a dynamic assignment of secondary structures that seem correlate with disordered states. In this study, we designed a single-task deep learning framework to predict IDP/IDR and SSP respectively; and multitask deep learning frameworks to allow quantitative predictions of IDP/IDR evidenced by the simultaneously predicted SSP. According to independent test results, single-task deep learning models improve the prediction performance of shallow models for SSP and IDP/IDR. Also, the prediction performance was further improved for IDP/IDR prediction when SSP prediction was simultaneously predicted in multitask models. With p53 as a use case, we demonstrate how predicted SSP is used to explain the IDP/IDR predictions for each functional region., (©2020 AMIA - All rights reserved.)

Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/T H 17 polarization. In murine NEC, pro-inflammatory type 3 NKp46 - RORγt + Tbet + innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46 + RORγt + ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.

Macrophage-expressed gene 1 [MPEG1/Perforin-2 (PRF2)] is an ancient metazoan protein belonging to the Membrane Attack Complex/Perforin (MACPF) branch of the MACPF/Cholesterol Dependent Cytolysin (CDC) superfamily of pore-forming proteins (PFPs). MACPF/CDC proteins are a large and extremely diverse superfamily that forms large transmembrane aqueous channels in target membranes. In humans, MACPFs have known roles in immunity and development. Like perforin (PRF) and the membrane attack complex (MAC), MPEG1 is also postulated to perform a role in immunity. Indeed, bioinformatic studies suggest that gene duplications of MPEG1 likely gave rise to PRF and MAC components. Studies reveal partial or complete loss of MPEG1 causes an increased susceptibility to microbial infection in both cells and animals. To this end, MPEG1 expression is upregulated in response to proinflammatory signals such as tumor necrosis factor α (TNFα) and lipopolysaccharides (LPS). Furthermore, germline mutations in MPEG1 have been identified in connection with recurrent pulmonary mycobacterial infections in humans. Structural studies on MPEG1 revealed that it can form oligomeric pre-pores and pores. Strikingly, the unusual domain arrangement within the MPEG1 architecture suggests a novel mechanism of pore formation that may have evolved to guard against unwanted lysis of the host cell. Collectively, the available data suggest that MPEG1 likely functions as an intracellular pore-forming immune effector. Herein, we review the current understanding of MPEG1 evolution, regulation, and function. Furthermore, recent structural studies of MPEG1 are discussed, including the proposed mechanisms of action for MPEG1 bactericidal activity. Lastly limitations, outstanding questions, and implications of MPEG1 models are explored in the context of the broader literature and in light of newly available structural data., (Copyright © 2020 Bayly-Jones, Pang, Spicer, Whisstock and Dunstone.)

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands., Competing Interests: Competing interestsThe authors declare no competing interests, (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background & Aims: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores., Methods: C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined., Results: hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination., Conclusions: We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses., Competing Interests: Declaration of Competing 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 © 2020 Elsevier Inc. All rights reserved.)

Background : CUB domain-containing protein 1 (CDCP1) is a cell surface receptor regulating key signalling pathways in malignant cells. CDCP1 has been proposed as a molecular target to abrogate oncogenic signalling pathways and specifically deliver anti-cancer agents to tumors. However, the development of CDCP1-targeting agents has been questioned by its frequent proteolytic processing which was thought to result in shedding of the CDCP1 extracellular domain limiting its targetability. In this study, we investigated the relevance of targeting CDCP1 in the context of pancreatic ductal adenocarcinoma (PDAC) and assess the impact of CDCP1 proteolysis on the effectiveness of CDCP1 targeting agents. Methods : The involvement of CDCP1 in PDAC progression was assessed by association analysis in several PDAC cohorts and the proteolytic processing of CDCP1 was evaluated in PDAC cell lines and patient-derived cells. The consequences of CDCP1 proteolysis on its targetability in PDAC cells was assessed using immunoprecipitation, immunostaining and biochemical assays. The involvement of CDCP1 in PDAC progression was examined by loss-of-function in vitro and in vivo experiments employing PDAC cells expressing intact or cleaved CDCP1. Finally, we generated antibody-based imaging and therapeutic agents targeting CDCP1 to demonstrate the feasibility of targeting this receptor for detection and treatment of PDAC tumors. Results : High CDCP1 expression in PDAC is significantly associated with poorer patient survival. In PDAC cells proteolysis of CDCP1 does not always result in the shedding of CDCP1-extracellular domain which can interact with membrane-bound CDCP1 allowing signal transduction between the different CDCP1-fragments. Targeting CDCP1 impairs PDAC cell functions and PDAC tumor growth independently of CDCP1 cleavage status. A CDCP1-targeting antibody is highly effective at delivering imaging radionuclides and cytotoxins to PDAC cells allowing specific detection of tumors by PET/CT imaging and superior anti-tumor effects compared to gemcitabine in in vivo models. Conclusion : Independent of its cleavage status, CDCP1 exerts oncogenic functions in PDAC and has significant potential to be targeted for improved radiological staging and treatment of this cancer. Its elevated expression by most PDAC tumors and lack of expression by normal pancreas and other major organs, suggest that targeting CDCP1 could benefit a significant proportion of PDAC patients. These data support the further development of CDCP1-targeting agents as personalizable tools for effective imaging and treatment of PDAC., Competing Interests: Competing Interests: Thomas Kryza, Tashbib Khan, Simon Puttick, Tahleesa Cuda, Elena I Deryugina, James P. Quigley, Yaowu He and John D. Hooper are inventors on a patent that includes monoclonal antibody 10D7 used in this study. No potential conflicts of interest were disclosed by the other authors., (© The author(s).)

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro , and tumor growth and metastasis in vivo . Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods : A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo , zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo . Results : Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo . Conclusions : These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Interleukin (IL)-37 is a member of the IL-1 family of cytokines. Although its broad anti-inflammatory properties are well described, the effects of IL-37 on inflammasome function remain poorly understood. Performing gene expression analyses, ASC oligomerization/speck assays and caspase-1 assays in bone marrow-derived macrophages (BMDM), and employing an in vivo endotoxemia model, we studied how IL-37 affects the expression and maturation of IL-1β and IL-18, inflammasome activation, and pyroptosis in detail. IL-37 inhibited IL-1β production by NLRP3 and AIM2 inflammasomes, and IL-18 production by the NLRP3 inflammasome. This inhibition was partially attributable to effects on gene expression: whereas IL-37 did not affect lipopolysaccharide (LPS)-induced mRNA expression of Il18 or inflammasome components, IL-37-transgenic BMDM displayed an up to 83% inhibition of baseline and LPS-stimulated Il1b compared to their wild-type counterparts. Importantly, we observed that IL-37 suppresses nigericin- and silica-induced ASC oligomerization/speck formation (a step in inflammasome activation and subsequent caspase-1 activation), and pyroptosis (-50%). In mice subjected to endotoxemia, IL-37 inhibited plasma IL-1β (-78% compared to wild-type animals) and IL-18 (-61%). Thus, our study adds suppression of inflammasome activity to the portfolio of anti-inflammatory pathways employed by IL-37, highlighting this cytokine as a potential tool for treating inflammasome-driven diseases.

The roles of proteolytic cleavage have been intensively investigated and discussed during the past two decades. This irreversible chemical process has been frequently reported to influence a number of crucial biological processes (BPs), such as cell cycle, protein regulation and inflammation. A number of advanced studies have been published aiming at deciphering the mechanisms of proteolytic cleavage. Given its significance and the large number of functionally enriched substrates targeted by specific proteases, many computational approaches have been established for accurate prediction of protease-specific substrates and their cleavage sites. Consequently, there is an urgent need to systematically assess the state-of-the-art computational approaches for protease-specific cleavage site prediction to further advance the existing methodologies and to improve the prediction performance. With this goal in mind, in this article, we carefully evaluated a total of 19 computational methods (including 8 scoring function-based methods and 11 machine learning-based methods) in terms of their underlying algorithm, calculated features, performance evaluation and software usability. Then, extensive independent tests were performed to assess the robustness and scalability of the reviewed methods using our carefully prepared independent test data sets with 3641 cleavage sites (specific to 10 proteases). The comparative experimental results demonstrate that PROSPERous is the most accurate generic method for predicting eight protease-specific cleavage sites, while GPS-CCD and LabCaS outperformed other predictors for calpain-specific cleavage sites. Based on our review, we then outlined some potential ways to improve the prediction performance and ease the computational burden by applying ensemble learning, deep learning, positive unlabeled learning and parallel and distributed computing techniques. We anticipate that our study will serve as a practical and useful guide for interested readers to further advance next-generation bioinformatics tools for protease-specific cleavage site prediction., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

VEK50 is a truncated peptide from a Streptococcal pyogenes surface human plasminogen (hPg) binding M-protein (PAM). VEK50 contains the full A-domain of PAM, which is responsible for its low nanomolar binding to hPg. The interaction of VEK50 with kringle 2, the PAM-binding domain in hPg (K2 hPg ), has been studied by high-resolution NMR spectroscopy. The data show that each VEK50 monomer in solution contains two tight binding sites for K2 hPg , one each in the a1- (RH1; R 17 H 18 ) and a2- (RH2; R 30 H 31 ) repeats within the A-domain of VEK50. Two mutant forms of VEK50, viz., VEK50[RH1/AA] (VEK50 ΔRH1 ) and VEK50[RH2/AA] (VEK50 ΔRH2 ), were designed by replacing each RH with AA, thus eliminating one of the K2 hPg binding sites within VEK50, and allowing separate study of each binding site. Using 13 C- and 15 N-labeled peptides, NMR-derived solution structures of VEK50 in its complex with K2 hPg were solved. We conclude that the A-domain of PAM can accommodate two molecules of K2 hPg docked within a short distance of each other, and the strength of the binding is slightly different for each site. The solution structure of the VEK50/K2 hPg , complex, which is a reductionist model of the PAM/hPg complex, provides insights for the binding mechanism of PAM to a host protein, a process that is critical to S. pyogenes virulence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

Plasminogen (Plg)-binding M protein (PAM) is a group A streptococcal cell surface receptor that is crucial for bacterial virulence. Previous studies revealed that, by binding to the kringle 2 (KR2) domain of host Plg, the pathogen attains a proteolytic microenvironment on the cell surface that facilitates its dissemination from the primary infection site. Each of the PAM molecules in their dimeric assembly consists of two Plg binding motifs (called the a1 and a2 repeats). To date, the molecular interactions between the a1 repeat and KR2 have been structurally characterized, whereas the role of the a2 repeat is less well defined. Here, we report the 1.7-Å x-ray crystal structure of KR2 in complex with a monomeric PAM peptide that contains both the a1 and a2 motifs. The structure reveals how the PAM peptide forms key interactions simultaneously with two KR2 via the high-affinity lysine isosteres within the a1a2 motifs. Further studies, through combined mutagenesis and functional characterization, show that a2 is a stronger KR2 binder than a1, suggesting that these two motifs may play discrete roles in mediating the final PAM-Plg assembly., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the P hoton I on E lectron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible., Competing Interests: SG, GB, GG, TJ, AH, MC, JW, RP, Ad No competing interests declared, (© 2019, Gorelick et al.)

Members of the membrane attack complex/perforin-like (MACPF) protein superfamily have long captured interest because of their unique ability to assemble into large oligomeric pores on the surfaces of cells. The best characterised of these act in vertebrate immunity where they function to deliver pro-apoptotic factors or induce the cytolysis and death of targeted cells. Less appreciated, however, is that rather than causing cell death, MACPF proteins have also evolved to control cellular signalling pathways and influence developmental programmes such as pattern formation and neurogenesis. Torso-like (Tsl) from the fruit fly Drosophila , for example, functions to localise the activity of a growth factor for patterning its embryonic termini. It remains unclear whether these developmental proteins employ an attenuated form of the classical MACPF lytic pore, or if they have evolved to function via alternative mechanisms of action. In this minireview, we examine the evidence that links pore-forming MACPF proteins to the control of growth factor and cytokine signalling. We will then attempt to reconcile how the MACPF domain may have been repurposed during evolution for developmental events rather than cell killing., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Plasminogen (Plg) is the zymogen form of the serine protease plasmin (Plm), and it plays a crucial role in fibrinolysis as well as wound healing, immunity, tissue remodeling and inflammation. Binding to the targets via the lysine-binding sites allows for Plg activation by plasminogen activators (PAs) present on the same target. Cellular uptake of fibrin degradation products leads to apoptosis, which represents one of the pathways for cross-talk between fibrinolysis and tissue remodeling. Therapeutic manipulation of Plm activity plays a vital role in the treatments of a range of diseases, whereas Plm inhibitors are used in trauma and surgeries as antifibrinolytic agents. Plm inhibitors are also used in conditions such as angioedema, menorrhagia and melasma. Here, we review the rationale for the further development of new Plm inhibitors, with a particular focus on the structural studies of the active site inhibitors of Plm. We compare the binding mode of different classes of inhibitors and comment on how it relates to their efficacy, as well as possible future developments., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses, and nonselective inhibition mechanisms. These drawbacks warrant the development of a new generation of highly potent and selective fibrinolysis inhibitors. Here, we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent ( K i = 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin, and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects.

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are two serine proteases that contribute to initiating fibrinolysis by activating plasminogen. uPA is also an important tumour-associated protease due to its role in extracellular matrix remodelling. Overexpression of uPA has been identified in several different cancers and uPA inhibition has been reported as a promising therapeutic strategy. Although several peptide-based uPA inhibitors have been developed, the extent to which uPA tolerates different tetrapeptide sequences that span the P1-P4 positions remains to be thoroughly explored. In this study, we screened a sequence-defined peptide aldehyde library against uPA and tPA. Preferred sequences from the library screen yielded potent inhibitors for uPA, led by Ac-GTAR-H (K i =18 nm), but not for tPA. Additionally, synthetic peptide substrates corresponding to preferred inhibitor sequences were cleaved with high catalytic efficiency by uPA but not by tPA. These findings provide new insights into the binding specificity of uPA and tPA and the relative activity of tetrapeptide inhibitors and substrates against these enzymes., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK-TcpK box complex reveals a binding mode centered on and around the β-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding.

Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.

The pore forming, Ca 2+ -dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca 2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca 2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin's cytotoxic function. These results revealed an unexpected role for the Ca 2+ -dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.

Phosphorylation is the most important type of protein post-translational modification. Accordingly, reliable identification of kinase-mediated phosphorylation has important implications for functional annotation of phosphorylated substrates and characterization of cellular signalling pathways. The local sequence context surrounding potential phosphorylation sites is considered to harbour the most relevant information for phosphorylation site prediction models. However, currently there is a lack of condensed vector representation for this important contextual information, despite the presence of varying residue-level features that can be constructed from sequence homology profiles, structural information, and physicochemical properties. To address this issue, we present PhosContext2vec which is a distributed representation of residue-level sequence contexts for potential phosphorylation sites and demonstrate its application in both general and kinase-specific phosphorylation site predictions. Benchmarking experiments indicate that PhosContext2vec could achieve promising predictive performance compared with several other existing methods for phosphorylation site prediction. We envisage that PhosContext2vec, as a new sequence context representation, can be used in combination with other informative residue-level features to improve the classification performance in a number of related bioinformatics tasks that require appropriate residue-level feature vector representation and extraction. The web server of PhosContext2vec is publicly available at http://phoscontext2vec.erc.monash.edu/.

In Drosophila , key developmental transitions are governed by the steroid hormone ecdysone. A number of neuropeptide-activated signaling pathways control ecdysone production in response to environmental signals, including the insulin signaling pathway, which regulates ecdysone production in response to nutrition. Here, we find that the Membrane Attack Complex/Perforin-like protein Torso-like, best characterized for its role in activating the Torso receptor tyrosine kinase in early embryo patterning, also regulates the insulin signaling pathway in Drosophila We previously reported that the small body size and developmental delay phenotypes of torso-like null mutants resemble those observed when insulin signaling is reduced. Here we report that, in addition to growth defects, torso-like mutants also display metabolic and nutritional plasticity phenotypes characteristic of mutants with impaired insulin signaling. We further find that in the absence of torso-like , the expression of insulin-like peptides is increased, as is their accumulation in insulin-producing cells. Finally, we show that Torso-like is a component of the hemolymph and that it is required in the prothoracic gland to control developmental timing and body size. Taken together, our data suggest that the secretion of Torso-like from the prothoracic gland influences the activity of insulin signaling throughout the body in Drosophila ., (Copyright © 2018 by the Genetics Society of America.)

Patterning of the Drosophila embryonic termini by the Torso (Tor) receptor pathway has long served as a valuable paradigm for understanding how receptor tyrosine kinase signaling is controlled. However, the mechanisms that underpin the control of Tor signaling remain to be fully understood. In particular, it is unclear how the Perforin-like protein Torso-like (Tsl) localizes Tor activity to the embryonic termini. To shed light on this, together with other aspects of Tor pathway function, we conducted a genome-wide screen to identify new pathway components that operate downstream of Tsl. Using a set of molecularly defined chromosomal deficiencies, we screened for suppressors of ligand-dependent Tor signaling induced by unrestricted Tsl expression. This approach yielded 59 genomic suppressor regions, 11 of which we mapped to the causative gene, and a further 29 that were mapped to <15 genes. Of the identified genes, six represent previously unknown regulators of embryonic Tor signaling. These include twins ( tws ), which encodes an integral subunit of the protein phosphatase 2A complex, and α-tubulin at 84B ( αTub84B ), a major constituent of the microtubule network, suggesting that these may play an important part in terminal patterning. Together, these data comprise a valuable resource for the discovery of new Tor pathway components. Many of these may also be required for other roles of Tor in development, such as in the larval prothoracic gland where Tor signaling controls the initiation of metamorphosis., (Copyright © 2018 Johns et al.)

While immunotherapy employing chimeric antigen receptor (CAR) T cells can be effective against a variety of tumor types, little is known about what happens within the tumor at an ultrastructural level during tumor regression. Here, we used transmission electron microscopy to investigate morphologic and cellular features of tumors responding to immunotherapy composed of adoptive transfer of dual-specific CAR T cells and a vaccine, supported by preconditioning irradiation and interleukin-2. Tumors responded rapidly, and large areas of cell death were apparent by 4 days after treatment. The pleomorphic and metabolically active nature of tumor cells and phagocytic activity of macrophages were apparent in electron microscopic images of tumors prior to treatment. Following treatment, morphologic features of various types of tumor cell death were observed, including apoptosis, paraptosis and necrosis. Large numbers of lipid droplets were evident in tumor cells undergoing apoptosis. Macrophages were the predominant leukocyte type infiltrating tumors before treatment. Macrophages decreased in frequency and number after treatment, whereas an increasing accumulation of neutrophils and T lymphocytes was observed following treatment. Phagocytic activity of macrophages and neutrophils was apparent, while T cells could be observed in close association with tumor cells with potential immunological synapses present. These observations highlight the cellular composition and ultrastructural appearance of tumors undergoing regression mediated by immunotherapy., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest associated with this study.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Cytotoxic lymphocytes play a key role in immune homeostasis through elimination of virally-infected and transformed target cells. They do this by employing the potent pore-forming protein, perforin, a molecule that permits cytotoxic proteases, such as granzyme B, to enter the target cell cytoplasm. The synergistic activities of perforin and granzymes bring about the destruction of target cells in a process that is now more clearly understood as a result of structural and cellular biology. These data are helping the development of new classes of immunosuppressive molecules for use in treating immune driven disease and in enhancing the success of transplant therapies. This review focuses on structural and biological aspects of perforin function., (Copyright © 2017. Published by Elsevier Ltd.)

Perforin is a highly cytotoxic pore-forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca 2+ -rich endoplasmic reticulum, remains unknown. Here, we show that N-linked glycosylation of the perforin C-terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C-terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C-terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post-translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule., (© 2017 The Authors.)

The rapid and orderly folding of epithelial tissue during developmental processes such as gastrulation requires the precise coordination of changes in cell shape. Here, we report that the perforin-like protein Torso-like (Tsl), the key extracellular determinant for Drosophila embryonic terminal patterning, also functions to control epithelial morphogenesis. We find that tsl null mutants display a ventral cuticular hole phenotype that is independent of the loss of terminal structures, and arises as a consequence of mesoderm invagination defects. We show that the holes are caused by uncoordinated constriction of ventral cell apices, resulting in the formation of an incomplete ventral furrow. Consistent with these data, we find that loss of tsl is sensitive to gene dosage of RhoGEF2 , a critical mediator of Rho1-dependent ventral cell shape changes during furrow formation, suggesting that Tsl may act in this pathway. In addition, loss of tsl strongly suppressed the effects of ectopic expression of Folded Gastrulation (Fog), a secreted protein that promotes apical constriction. Taken together, our data suggest that Tsl controls Rho1-mediated apical constriction via Fog. Therefore, we propose that Tsl regulates extracellular Fog activity to synchronize cell shape changes and coordinate ventral morphogenesis in Drosophila Identifying the Tsl-mediated event that is common to both terminal patterning and morphogenesis will be valuable for our understanding of the extracellular control of developmental signaling by perforin-like proteins., (Copyright © 2017 by the Genetics Society of America.)