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Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI–light-harvesting complex I monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH and thus partially overlaps with the surface area that would bind one of the light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI–light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

GNG5 is suggested to exert a critical effect on tumor development in human beings; however, its function and related mechanism within breast cancer (BC) are still unclear. In this regard, the present work focused on identifying and evaluating GNG5's function and revealing its possible molecular mechanism. Subcutaneous tumorigenesis model of nude mice and in-vitro cell model was established. The relationship between GNG5 expression and BC was studied through knockdown and overexpression experiments. The proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of liver cancer cell lines overexpressing or silencing GNG5 were detected. Furthermore, the pathway mechanism of GNG5 was evaluated at the molecular level and was performed to further verify the possible targets and mechanisms of action. In comparison with that in normal tissue, GNG5 level within BC tissue was higher. In addition, GNG5 overexpression stimulated BC cell proliferation, invasion, migration and EMT. BC cells with reduced GNG5 expression exhibited significant decreases in glucose uptake, lactate levels, and ATP concentrations. In addition, GNG5 knockdown inhibited Wnt/β-catenin signaling. This study indicates that GNG5 may generate a vital function in BC. The results of the current work demonstrated GNG5's effect on BC pathological process, also providing a reference for developing new targeted therapies for BC.

Filoviruses, including Ebola virus, pose an increasing threat to the public health. Although two therapeutic monoclonal antibodies have been approved to treat the Ebola virus disease

Cyclophosphamide is a well-known anticancer agent acting by means of DNA alkylation. Associated with its tumor selectivity, it also possesses a wide spectrum of toxicities. As the requirement of metabolic activation before cyclophosphamide exerts either its therapeutic or toxic effects is well recognized, research aiming at elucidating the pathways that lead to the activation of this drug is of key importance. This has created the necessity for developing an effective analytical method for detecting cyclophosphamide and its breakdown products. In this paper, an Acquity TQ tandem quadrupole mass spectrometer equipped with electrospray ionization in positive-ion mode was employed for detecting cyclophosphamide in its protonated form. The full-scan mass spectrum of cyclophosphamide shows two ion clusters displaying the characteristic isotopic pattern of two chlorine atoms and assigned as sodiated cyclophosphamide, [CP + Na]

Voltage-gated potassium (Kv) channels each comprise four pore-forming α-subunits that orchestrate essential duties such as voltage sensing and K+ selectivity and conductance. In vivo, however, Kv channels also incorporate regulatory subunits—some Kv channel specific, others more general modifiers of protein folding, trafficking, and function. Understanding all the above is essential for a complete picture of the role of Kv channels in physiology and disease.

We report a joint experimental-theoretical study of the never reported before structure and infrared spectra of gas phase monohydrated nicotine (NIC) and nornicotine (NOR) and use them to assign their protonation sites. NIC's biological activity is strongly affected by its protonation site, namely, the pyrrolidine (Pyrro-NICH

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565–587, called CaM1) decreases the open probability of CNG channels at elevated Ca2+ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120–1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447).

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease.

The mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients(1,2). Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L, SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors Sestrin2 and CASTOR1, respectively(3–5). Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function, and structure are unknown. Here, we used electron cryomicroscopy to determine the three-dimensional structure of the human GATOR2 complex. We find that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS, and two WDR59 subunits circularized via two distinct types of junctions involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane coating complexes(6). The scaffold orients the WD40 β-propeller dimers, which mediate interactions with Sestrin2, CASTOR1, and GATOR1. Our work reveals the structure of an essential component of the nutrient sensing machinery and provides a foundation for understanding GATOR2 function within the mTORC1 pathway.

Unexpected non-apparent and hidden allosteric-binding sites are non-classical and non-apparent allosteric centers in 3-D X-ray protein structures until orthosteric or allosteric ligands bind to them. The orthosteric center of one protomer that modulates binding centers of the other protomers within an oligomer is also an unexpected allosteric site. Furthermore, another partner protein can also produce these effects, acting as an unexpected allosteric modulator.This review summarizes both classical and non-classical allosterism. The authors focus on G protein-coupled receptor (GPCR) oligomers as a paradigm of allosteric molecules. Moreover, they show several examples of unexpected allosteric sites such as hidden allosteric sites in a protomer that appear after the interaction with other molecules and the allosterism exerted between orthosteric sites within GPCR oligomer, emphasizing on the allosteric modulations that can occur between binding sites.The study of these new non-classical allosteric sites will expand the diversity of allosteric control on the function of orthosteric sites within proteins, whether GPCRs or other receptors, enzymes, or transporters. Moreover, the design of new drugs targeting these hidden allosteric sites or already known orthosteric sites acting as allosteric sites in protein homo- or hetero-oligomers will increase the therapeutic potential of allosterism.

Streptococcus oralis is a member of the mitis group of oral streptococci and an early colonizer in dental plaque biofilm, a major cause of periodontal disease, dental caries, and other oral infections. S. oralis promotes biofilm growth by coaggregating in a mutualistic partnership with other early colonizers such as Actinomyces oris. For this cell-to-cell interaction, A. oris is known to use its sortase-dependent pilus (type 2), but whether S. oralis uses its PI-2 (pilus islet 2) pilus is still to be determined. The PI-2 pilus is predicted to have a heterodimeric structure consisting of two different protein subunits with their own location and function: the tip PitA pilin for adhesion and the backbone PitB pilin for length. Thus far, structural information remains incomplete about the role of PI-2 pili in the mutualistic mechanism between S. oralis and A. oris. We now report on the crystal structure analysis of PitA and PitB using X-ray crystallography, small-angle X-ray scattering, and molecular docking studies. Accordingly, we propose a structural model for the PI-2 pilus, wherein repeating PitB subunits are arranged head-to-tail to form the long backbone structure with PitA on the outer tip. By performing both in vitro and in vivo experiments, we examined the role played by PitA in mediating the mutualistic interaction between S. oralis and A. oris, which appears to involve the coaggregation factor CafA. We also reveal that the galactose monosaccharide is a conceivable ligand for PitA and thereby might be used to inhibit coaggregation and control oral biofilm development. DATABASE: Structural coordinates for the PitA fragment, PitA fragment TbXO4 derivative, full-length PitA, and PitB from S. oralis have been deposited at the Protein Data Bank as 7VCR, 7W7I, 7VCN, 7W6B, and 7W7I, respectively. Streptococcus pneumoniae PitB coordinates have been deposited as 7F7Y.

Respiratory Complex I is the site of a large fraction of the mutations that appear to cause mitochondrial disease. Seven of its subunits are mitochondrially encoded, and therefore, such mutants are particularly difficult to construct in cell-culture model systems. We have selected 13 human clinical mutations found in ND2, ND3, ND4, ND4L, ND5 and ND6 that are generally found at subunit interfaces, and not in critical residues. These mutations have been modeled in E. coli subunits of Complex I, nuoN, nuoA, nuoM, nuoK, nuoL, and nuoJ, respectively. All mutants were expressed from a plasmid encoding the entire nuo operon, and membrane vesicles were analyzed for deamino-NADH oxidase activity, and proton translocation activity. ND5 mutants were also analyzed using a time-delayed expression system, recently described by this lab. Other mutants were analyzed for the ability to associate in subcomplexes, after expression of subsets of the genes. For most mutants there was a positive correlation between those that were previously determined to be pathogenic, or likely to be pathogenic, and those that we found with compromised Complex I activity or subunit interactions in E. coli. In conclusion, this approach provides another way to explore the deleterious effects of human mitochondrial mutations, and it can contribute to molecular understanding of such mutations.

Background Group B Streptococcus (GBS) transmission during pregnancy causes preterm labor, stillbirths, fetal injury, or neonatal infections. Rates of adult infections are also rising. The GBS-NN vaccine, engineered by fusing N-terminal domains of GBS Alpha C and Rib proteins, is safe in healthy, nonpregnant women, but further assessment is needed for use during pregnancy. Here, we tested GBS-NN vaccine efficacy using mouse models that recapitulate human GBS infection outcomes. Methods Following administration of GBS-NN vaccine or adjuvant, antibody profiles were compared by ELISA. Vaccine efficacy was examined by comparing infection outcomes in GBS-NN vaccinated versus adjuvant controls during systemic and pregnancy-associated infections, and during intranasal infection of neonatal mice following maternal vaccination. Results Vaccinated mice had higher GBS-NN–specific IgG titers versus controls. These antibodies bound alpha C and Rib on GBS clinical isolates. Fewer GBS were recovered from systemically challenged vaccinated mice versus controls. Although vaccination did not eliminate GBS during ascending infection in pregnancy, vaccinated dams experienced fewer in utero fetal deaths. Additionally, maternal vaccination prolonged neonatal survival following intranasal GBS challenge. Conclusions These findings demonstrate GBS-NN vaccine efficacy in murine systemic and perinatal GBS infections and suggest that maternal vaccination facilitates the transfer of protective antibodies to neonates.

Many aspects of plant photoperception are mediated by the phytochrome (Phy) family of bilin-containing photoreceptors that reversibly interconvert between inactive Pr and active Pfr conformers(1,2). Despite extensive biochemical studies, full understanding of plant Phy signalling has remained unclear due to the absence of relevant 3D models. Here we report a cryo-electron microscopy structure of Arabidopsis PhyB in the Pr state that reveals a topologically complex dimeric organization that is substantially distinct from its prokaryotic relatives. Instead of an anticipated parallel architecture, the C-terminal histidine-kinase-related domains (HKRDs) associate head-to-head, whereas the N-terminal photosensory regions associate head-to-tail to form a parallelogram-shaped platform with near two-fold symmetry. The platform is internally linked by the second of two internal Per/Arnt/Sim domains that binds to the photosensory module of the opposing protomer and a preceding ‘modulator’ loop that assembles tightly with the photosensory module of its own protomer. Both connections accelerate the thermal reversion of Pfr back to Pr, consistent with an inverse relationship between dimer assembly and Pfr stability. Lopsided contacts between the HKRDs and the platform create profound asymmetry to PhyB that might imbue distinct signalling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate-sensitive surface for conformation-dependent interactions between plant Phy photoreceptors and their signalling partners.

Mistletoes (Viscum) and close relatives are unique among flowering plants in having a drastically altered electron transport chain. Lack of complex I genes has previously been reported for the mitochondrial genome, and here we report an almost complete absence of nuclear-encoded complex I genes in the transcriptome of Viscum album. Compared to Arabidopsis with approximately 40 nuclear complex I genes, we recover only transcripts of two dual-function genes: gamma carbonic anhydrase and L-galactono-1,4-lactone dehydrogenase. The complement of genes belonging to complexes II-V of the oxidative phosphorylation pathway appears to be in accordance with other vascular plants. Additionally, transcripts encoding alternative NAD(P)H dehydrogenases and alternative oxidase were found. Despite sequence divergence, structural modeling suggests that the encoded proteins are structurally conserved. Complex I loss is a special feature in Viscum species and relatives, as all other parasitic flowering plants investigated to date seem to have a complete OXPHOS system. Hence, Viscum offers a unique system for specifically investigating molecular consequences of complex I absence, such as the role of complex I subunits involved in secondary functions.

Hyperglycemia, which occurs under the diabetic conditions, induces serious diabetic complications. Diabetic encephalopathy has been defined as one of the major complications of diabetes, and is characterized by neurochemical and neurodegenerative changes. However, little is known about the effect of long-term exposure to high glucose on neuronal cells. In the present study, we showed that exposure to glutamate (100 mM) for 7 days induced toxicity in primary cortical neurons using the MTT assay. Additionally, high glucose increased the sensitivity of AMPA- or NMDA-induced neurotoxicity, and decreased extracellular glutamate levels in primary cortical neurons. In Western blot analyses, the protein levels of the GluA1 and GluA2 subunits of the AMPA receptor as well as synaptophysin in neurons treated with high glucose were significantly increased compared with the control (25 mM glucose). Therefore, long-term exposure to high glucose induced neuronal death through the disruption of glutamate homeostasis.

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA control of pho1 mRNA synthesis is influenced by inositol pyrophosphate (IPP) kinase Asp1, deletion of which results in pho1 hyper-repression. A forward genetic screen for ADS (Asp1 Deletion Suppressor) mutations identified the 14–3–3 protein Rad24 as a governor of phosphate homeostasis. Production of full-length interfering prt lncRNA was squelched in rad24Δ cells, concomitant with increased production of pho1 mRNA and increased Pho1 activity, while shorter precociously terminated non-interfering prt transcripts persisted. Epistasis analysis showed that pho1 de-repression by rad24Δ depends on: (i) 3′-processing and transcription termination factors CPF, Pin1, and Rhn1; and (ii) Threonine-4 of the Pol2 CTD. Combining rad24Δ with the IPP pyrophosphatase-dead asp1-H397A allele caused a severe synthetic growth defect that was ameliorated by loss-of-function mutations in CPF, Pin1, and Rhn1, and by CTD phospho-site mutations T4A and Y1F. Rad24 function in repressing pho1 was effaced by mutation of its phosphate-binding pocket. Our findings instate a new role for a 14–3–3 protein as an antagonist of precocious RNA 3′-processing/termination.

In rod photoreceptors of the retina, the cyclic nucleotide-gated (CNG) channel is composed of three CNGA and one CNGB subunits, and it closes in response to light activation to generate an electrical signal that is conveyed to the brain. Here we report the cryo-EM structure of the closed state of the native rod CNG channel isolated from bovine retina. The structure reveals differences between CNGA1 and CNGB1 subunits. Three CNGA1 subunits are tethered at their C terminus by a coiled-coil region. The C-helix in the cyclic nucleotide-binding domain of CNGB1 features a different orientation from that in the three CNGA1 subunits. The arginine residue R994 of CNGB1 reaches into the ionic pathway and blocks the pore, thus introducing an additional gate, which is different from the central hydrophobic gate known from homomeric CNGA channels. These results address the long-standing question of how CNGB1 subunits contribute to the function of CNG channels in visual and olfactory neurons.

The anticodon stem of initiator tRNA (i-tRNA) possesses the characteristic three consecutive GC base pairs (G29:C41, G30:C40, and G31:C39 abbreviated as GC/GC/GC or 3GC pairs) crucial to commencing translation. To understand the importance of this highly conserved element, we isolated two fast-growing suppressors of Escherichia coli sustained solely on an unconventional i-tRNA (i-tRNA

The SARS-CoV-2 virus has been known to gain entry into the host cell through the spike protein that binds to the host ACE2 cell surface protein. However, the role of the putative sugar-binding sites in the spike protein has remained unclear. We provide a comprehensive in silico outlook into the infection initiation wherein the virus first recognizes the sialosides on the cell via its S1A domain of the spike protein as it surfs over the cell surface. This facilitates the subsequent interaction with the cellular glycosaminoglycans through the S1B domain of the spike protein as it binds to the ACE2 receptor. The unique coadaptation to recognize both the host protein and the cell-surface carbohydrate receptors provides an additional coupling mechanism for efficient viral attachment and infection.

NMDAR (N-methyl-d-aspartate receptor) consisted of GluN1 and GluN2, and/or GluN3 subunits. As the obligatory subunit of NMDAR, GluN1 contains variant N-terminal domain (NTD) and C-terminal domain (CTD). The CTD contains allosteric signal and mediates the metabotropic function of NMDAR, which has been confirmed by previous studies. However, the allosteric signaling mechanism of GluN1 CTD has not been studied. In our study, we found that GluN1 CTD could bind to the lipid bilayers and affect the antigen epitope of GluN1 C-terminal antibody, suggesting that membrane binding may determine the allosteric signal of GluN1 CTD. In addition, we discovered that the membrane binding of GluN1 CTD could be regulated by the phosphorylation of GluN1 CTD C1 region.

Not your typical GPCR Among the large family of G protein–coupled receptors (GPCRs) are many orphans, so called because their signaling reactions remain poorly understood. Among these is GPR158 which is highly expressed in the nervous system and implicated in processes from cognition to memory to mood. Patil et al . determined a high-resolution structure of GPR158 alone and bound to a regulator of G protein signaling (RGS) complex. GPR158 has an unusual dimerization mode with an extensive interaction interface that locks it in a conformation that likely prevents G protein activation. RGS binds to the homodimer at a site that substantially overlaps the surface that binds G proteins, again preventing canonical G protein signaling. Binding of a ligand to the extracellular domain may regulate signaling through the RGS complex. —VV

Integrator and protein phosphatase 2A (PP2A) form a complex that dephosphorylates paused RNA polymerase II (Pol II), cleaves the nascent RNA, and terminates transcription. We report the structure of the pretermination complex containing the human Integrator-PP2A complex bound to paused Pol II. Integrator binds Pol II and the pausing factors DSIF and NELF to exclude binding of the elongation factors SPT6 and PAF1 complex. Integrator also binds the C-terminal domain of Pol II and positions PP2A to counteract Pol II phosphorylation and elongation. The Integrator endonuclease docks to the RNA exit site and opens to cleave nascent RNA about 20 nucleotides from the Pol II active site. Integrator does not bind the DNA clamps formed by Pol II and DSIF, enabling release of DNA and transcription termination.

Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside homology model guided design, to develop chemical biology approaches for the study of BRD8 inhibition, and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

Ribosome profiling (riboseq) has opened the possibilities for the genome-wide studies of translation in all living organisms. This method is based on deep sequencing of mRNA fragments protected by the ribosomes from hydrolysis by ribonucleases, the so-called ribosomal footprints (RFPs). Ribosomal profiling together with RNA sequencing allows not only to identify with a reasonable accuracy translated reading frames in the transcriptome, but also to track changes in gene expression in response to various stimuli. Notably, ribosomal profiling in its classical version has certain limitations. The size of the selected mRNA fragments is 25-35 nts, while RFPs of other sizes are usually omitted from analysis. Also, ribosomal profiling "averages" the data from all ribosomes and does not allow to study specific ribosomal complexes associated with particular translation factors. However, recently developed modifications of ribosomal profiling provide answers to a number of questions. Thus, it has become possible to analyze not only elongating, but also scanning and reinitiating ribosomes, to study events associated with the collision of ribosomes during mRNA translation, to discover new ways of cotranslational assembly of multisubunit protein complexes during translation, and to selectively isolate ribosomal complexes associated with certain protein factors. New data obtained using these modified approaches provide a better understanding of the mechanisms of translation regulation and the functional roles of translational apparatus components.

Despite its similarity to protein biosynthesis in bacteria, translation in the mitochondria of modern eukaryotes has several unique features, such as the necessity for coordination of translation of mitochondrial mRNAs encoding proteins of the electron transport chain complexes with translation of other protein components of these complexes in the cytosol. In the mitochondria of baker’s yeast Saccharomyces cerevisiae, this coordination is carried out by a system of translational activators that predominantly interact with the 5′-untranslated regions of mitochondrial mRNAs. No such system has been found in human mitochondria, except a single identified translational activator, TACO1. Here, we studied the role of the ZMYND17 gene, an ortholog of the yeast gene for the translational activator Mss51p, on the mitochondrial translation in human cells. Deletion of the ZMYND17 gene did not affect translation in the mitochondria, but led to the decrease in the cytochrome c oxidase activity and increase in the amount of free F1 subunit of ATP synthase. We also investigated the evolutionary history of Mss51p and ZMYND17 and suggested a possible mechanism for the divergence of functions of these orthologous proteins.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 μM; LRL-TP-94: 2.9 μM; and LRL-TP-101: 35.3 μM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 μM in extracellular promastigote assay and 34.0, 53.7, and 11.4 μM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

BackgroundIn response to SARS-CoV-2 mutations and waning antibody levels after two-dose inactivated vaccines, we assessed whether a third dose of recombinant protein subunit vaccine (ZF2001) boosts immune responses.MethodsAn open-label single-center non-random trial was conducted on people aged 18 years and above at five sites in China. All participants received a two-dose inactivated vaccine (CoronaVac) as their prime doses within 3–9 months of the trial. Primary outcomes were safety and immunogenicity, primarily the geometric mean titers (GMTs) of neutralizing antibodies to live wildtype SARS-CoV-2.ResultsA total of 480 participants (median age, 51; range 21–84 years) previously vaccinated with two-dose CoronaVac received a third booster dose of ZF2001 3–4, 5–6, or 7–9-months later. The overall incidence of adverse reactions within 30 days after vaccination was 5.83% (28/480). No serious adverse reactions were reported after the third dose of ZF2001. GMTs in the 3–4-, 5–6-, and 7–9-month groups before vaccination were 3.96, 4.60, and 3.78, respectively. On Day 14, GMTs increased to 33.06, 47.51, and 44.12, respectively. After the booster, GMTs showed no significant difference among the three prime-boost interval groups (all P>0.05). Additionally, GMTs in older adults were lower than those in younger adults on Day 14 for the three groups (P=0.0005, PConclusionHeterologous boosting with ZF2001 was safe and immunogenic, and prime-boost intervals did not affect the immune response. The immune response was weaker in older than younger adults.

Confronted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as Delta and Omicron, with high infectivity and immune evasion capacity, vaccination remains the most effective tool to prevent infection and severe illness. However, heterologous vaccination of mRNA vaccines primed with protein subunit vaccines had not been evaluated before the current study. Since subunit vaccine MVC-COV1901 (MVC) has been granted emergency use authorization in Taiwan, in this study, we explored the humoral and cellular immune responses to additional third (2× MVC/Mod) and fourth (2× MVC/2× Mod) doses of mRNA-1273 (Mod) after priming with two doses of subunit vaccine MVC against the emerging variants. We found a 12.3- to 16.1-fold increase in antibodies targeting the receptor binding domain (RBD) of the Delta variant with 2× MVC/Mod compared to two doses of MVC (2× MVC) or AZD1222 (2× AZ) regimens and a 26- to 32.2-fold improvement in neutralizing potency against the Omicron variant (BA.1). Besides, the numbers of gamma interferon (IFN-γ)-secreting T cells induced by 2× MVC/Mod were also elevated 3.5-fold and 3.7- to 4.3-fold for the wild type and Delta variant. However, boosting with a fourth dose of Mod (2× MVC/2× Mod) after the 2× MVC/Mod regimen failed to significantly improve the immune responses. Moreover, all vaccination schedules showed reduced neutralizing activity against the Omicron variant. Collectively, our results suggested that the third or fourth dose booster vaccination with mRNA vaccine after priming with two doses of protein subunit vaccine could elicit stronger humoral and cellular immune responses. These findings could provide a future global heterologous boosting strategy against COVID-19.

Under the background of the severe human health and world economic burden caused by COVID-19, the attenuation of vaccine protection efficacy, and the prevalence and immune escape of emerging variants of concern (VOCs), the third dose of booster immunization has been put on the agenda. Systems biology approaches can help us gain new perspectives on the characterization of immune responses and the identification of factors underlying vaccine-induced immune efficacy. We analyzed the antibody signature and transcriptional responses of participants vaccinated with COVID-19 inactivated vaccine and protein subunit vaccine as a third booster dose. The results from the antibody indicated that the third booster dose was effective, and that heterologous vaccination with the protein subunit vaccine as a booster dose induced stronger humoral immune responses than the homologous vaccination with inactivated vaccine, and might be more effective against VOCs. In transcriptomic analysis, protein subunit vaccine induced more differentially expressed genes that were significantly associated with many important innate immune pathways. Both the homologous and heterologous boosters could increase the effectiveness against COVID-19, and compared with the inactivated vaccine, the protein subunit vaccine, mediated a stronger humoral immune response and had a more significant correlation with the innate immune function module, which provided certain data support for the third booster immunization strategy.

4-aminobenzoic acid (4ABA) is a model scaffold for studying solvent-mediated proton transfer. Although protonation at the carboxylic group ( O-protomer) is energetically favored in the gas phase, the N-protomer, where the proton remains on the amino group, can be kinetically trapped by electrospray ionization of 4ABA in an aprotic solvent such as acetonitrile. Here, we report the formation of the hydrated deuterium isotopologues of the N-protomers, RND3+·(H2O) n=1–3, (R = C6H4COOD), which are generated by condensing water molecules onto the bare N-protomers in a liquid nitrogen cooled, radiofrequency octopole ion trap at 80 K. The product clusters are then transferred to a 20 K cryogenic ion trap where they are tagged with weakly bound D2 molecules. The structures of these clusters are determined by analysis of their vibrational patterns, obtained by resonant IR photodissociation. The resulting patterns confirm that the metastable N-protomer configuration remains intact even when warmed by the sequential condensation of water molecules. The attachment of H2O molecules onto the RND3+ head group also affords the opportunity to explore the possibility of H/D exchange between the acid scaffold and the proximal water network. The spectroscopic results establish that although the RND3+·(H2O) n=1,2 clusters are formed without H/D exchange, the n = 3 cluster exhibits about 10% H/D exchange as evidenced by the appearance of the telltale HOD bands. The site of exchange on the acid is determined to be the acidic OH group by the emergence of the OH stretching fundamental in the –COOH motif.

The Ebola virus has caused outbreaks in Central and West Africa, with high rates of morbidity and mortality. Clinical trials of recombinant virally vectored vaccines did not explicitly include pregnant or nursing women, resulting in a gap in knowledge of vaccine-elicited maternal antibody and its potential transfer. The role of maternal antibody in Ebola virus disease and vaccination remains understudied. Here, we demonstrate that a protein subunit vaccine can elicit robust humoral responses in pregnant mice, which are transferred to pups in breastmilk. These findings indicate that an intramuscular protein subunit vaccine may elicit Ebola-specific IgG capable of being transferred across the placenta as well as into the breastmilk. We have previously shown protective efficacy with these vaccines in non-human primates, offering a potential safe and practical alternative to recombinant virally vectored vaccines for pregnant and nursing women in Ebola endemic regions.

The SARS-CoV-2 prefusion spike protein is characterized by a high degree of flexibility and temporal transformations associated with its multifunctional behavior. In this study, we have examined the dynamics of the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein in detail. Its primary, binding subdomain with human Angiotensin Covering Enzyme II includes a highly conspicuous flap or loop that is part of a beta hairpin loop structural motif. Dynamic details of the RBD obtained through RMSF and Order Parameter calculations are consistent with structural details including the stability of “glue” points or dominant interaction energy residues of the RBD in the Up and Down states with its neighboring N-terminal domain (NTD) protomer. The RBD flap in the Up state protomer periodically obstructs the binding site on an approximate 70 nanosecond time interval and is reminiscent of an HIV-1 protease polypeptide flap that opens and closes to modulate that enzymes activity. No claim is made here regarding the possible modulating role of the flap; however, the flap may be a potential site for therapeutic targeting aimed at keeping it in the closed state, as previously demonstrated in the inhibition of the HIV-1 protease polypeptide. The RBD primary binding subdomain is further shown to have not only similar dynamics but, also, an approximate 30% sequence similarity to the HIV-1 protease polypeptide.

Neural communication and modulation are complex processes. Ionotropic glutamate receptors (iGluRs) significantly contribute to mediating the fast-excitatory branch of neurotransmission in the mammalian brain. Kainate receptors (KARs), a subfamily of the iGluRs, act as modulators of the neuronal circuitry by playing important roles at both the post- and presynaptic sites of specific neurons. The functional tetrameric receptors are formed by two different gene families, low agonist affinity (GluK1-GluK3) and high agonist affinity (GluK4-GluK5) subunits. These receptors garnered attention in the past three decades, and since then, much work has been done to understand their localization, interactome, physiological functions, and regulation. Cloning of the receptor subunits (GluK1-GluK5) in the early 1990s led to recombinant expression of kainate receptors in heterologous systems. This facilitated understanding of the functional differences between subunit combinations, splice variants, trafficking, and drug discovery. Structural studies of individual domains and recent full-length homomeric and heteromeric kainate receptors have revealed unique functional mechanisms, which have answered several long-standing questions in the field of kainate receptor biology. In this chapter, we review the current understanding of kainate receptors and associated disorders.

Photosystem I (PSI) is a protein complex functioning in light-induced charge separation, electron transfer, and reduction reactions of ferredoxin in photosynthesis, which finally results in the reduction of NAD(P)

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms −1 ) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein–phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.

The recently emerged Omicron (B.1.1.529) variant has rapidly surpassed Delta to become the predominant circulating SARS-CoV-2 variant, given the higher transmissibility rate and immune escape ability, resulting in breakthrough infections in vaccinated individuals. A new generation of SARS-CoV-2 vaccines targeting the Omicron variant are urgently needed. Here, we developed a subunit vaccine named RBD-HR/trimer by directly linking the sequence of RBD derived from the Delta variant (containing L452R and T478K) and HR1 and HR2 in SARS-CoV-2 S2 subunit in a tandem manner, which can self-assemble into a trimer. In multiple animal models, vaccination of RBD-HR/trimer formulated with MF59-like oil-in-water adjuvant elicited sustained humoral immune response with high levels of broad-spectrum neutralizing antibodies against Omicron variants, also inducing a strong T cell immune response in vivo. In addition, our RBD-HR/trimer vaccine showed a strong boosting effect against Omicron variants after two doses of mRNA vaccines, featuring its capacity to be used in a prime-boost regimen. In mice and non-human primates, RBD-HR/trimer vaccination could confer a complete protection against live virus challenge of Omicron and Delta variants. The results qualified RBD-HR/trimer vaccine as a promising next-generation vaccine candidate for prevention of SARS-CoV-2, which deserved further evaluation in clinical trials.

Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here, we use deep network hallucination to generate a wide range of symmetric protein homo-oligomers given only a specification of the number of protomers and the protomer length. Crystal structures of seven designs are very similar to the computational models (median root mean square deviation: 0.6 angstroms), as are three cryo–electron microscopy structures of giant 10-nanometer rings with up to 1550 residues and C 33 symmetry; all differ considerably from previously solved structures. Our results highlight the rich diversity of new protein structures that can be generated using deep learning and pave the way for the design of increasingly complex components for nanomachines and biomaterials.

Safeguarding protein complex assembly The assembly of multiprotein complexes inside the cell requires each subunit to be produced at a defined level relative to its partners. Imbalances in subunit synthesis are inevitable, necessitating the elimination of unassembled intermediates. Zavodszky et al . found that a ubiquitin ligase called HERC1 is responsible for marking certain assembly intermediates of the proteasome for degradation. HERC1 finds these intermediates by recognizing a proteasome assembly factor that normally dissociates when assembly is complete. A point mutation in HERC1 that impairs its ability to recognize proteasome assembly intermediates causes neurodegeneration in mice, highlighting the importance of this quality control pathway. —SMH

For CASP14, we developed deep learning-based methods for predicting homo-oligomeric and hetero-oligomeric contacts and used them for oligomer modeling. To build structure models, we developed an oligomer structure generation method that utilizes predicted interchain contacts to guide iterative restrained minimization from random backbone structures. We supplemented this gradient-based fold-and-dock method with template-based and ab initio docking approaches using deep learning-based subunit predictions on 29 assembly targets. These methods produced oligomer models with summed Z-scores 5.5 units higher than the next best group, with the fold-and-dock method having the best relative performance. Over the eight targets for which this method was used, the best of the five submitted models had average oligomer TM-score of 0.71 (average oligomer TM-score of the next best group: 0.64), and explicit modeling of inter-subunit interactions improved modeling of six out of 40 individual domains (ΔGDT-TS > 2.0).

The Rel proteins of the NF-κB complex comprise one of the most investigated transcription factor families, forming a variety of hetero- or homodimers. Nevertheless, very little is known about the fundamental kinetics of NF-κB complex assembly, or the inter-conversion potential of dimerised Rel subunits. Here, we examined an unexplored aspect of NF-κB dynamics, focusing on the dissociation and reassociation of the canonical p50 and p65 Rel subunits and their ability to form new hetero- or homodimers. We employed a soluble expression system to enable the facile production of NF-κB Rel subunits, and verified these proteins display canonical NF-κB nucleic acid binding properties. Using a combination of biophysical techniques, we demonstrated that, at physiological temperatures, homodimeric Rel complexes routinely exchange subunits with a half-life of less than 10 min. In contrast, we found a dramatic preference for the formation of the p50/p65 heterodimer, which demonstrated a kinetic stability of at least an order of magnitude greater than either homodimer. These results suggest that specific DNA targets of either the p50 or p65 homodimers can only be targeted when these subunits are expressed exclusively, or with the intervention of additional post-translational modifications. Together, this work implies a new model of how cells can modulate NF-κB activity by fine-tuning the relative proportions of the p50 and p65 proteins, as well as their time of expression. This work thus provides a new quantitative interpretation of Rel dimer distribution in the cell, particularly for those who are developing mathematical models of NF-κB activity.

Data mining on a public domain detected eight potential transcripts which were upregulated in advanced UBUCs, suggesting that they may take part in UC development or/and progression. Retrospectively, immunohistochemistry along with H-score recording was carried out to evaluate the GNB4 protein levels on tissues from UC patients. Correlations between GNB4 H-score and imperative clinicopathological factors, as well as the implication of GNB4 protein level on disease-specific and metastasis-free survivals were assessed. In UTUCs (n = 340) and UBUCs (n = 295), 170 (50.0%) and 148 (50.0%) cases, respectively, were identified to be of high GNB4 expression. The GNB4 protein levels were correlated to numerous clinicopathological features and patients' survivals. Upregulation of the GNB4 protein was significantly associated with primary tumor, nodal metastasis, histological grade, vascular invasion and mitotic rate. High GNB4 protein levels independently and significantly predicted poor disease-specific and metastasis-free in UTUC and UBUC, respectively. Ingenuity pathway analysis furthermore showed that multiple signaling pathways were enriched including 'Communication between Innate and Adaptive Immune Cells' and 'NFκB Signaling'. Our findings demonstrated that the upregulation of the GNB4 protein is an independent unfavorable prognosticator in UC. High GNB4 gene expression plays an important role in UC progression.

Proteins perform their functions by interacting with other biomolecules. For these interactions, proteins often form homo- or hetero-oligomers as well. Thus, oligomer protein structures provide important clues regarding the biological roles of proteins. To this end, computational prediction of oligomer structures may be a useful tool in the absence of experimentally resolved structures. Here, we describe our server and human-expert methods used to predict oligomer structures in the CASP14 experiment. Examples are provided for cases in which manual domain-splitting led to improved oligomeric domain structures by ab initio docking, automated oligomer structure refinement led to improved subunit orientation and terminal structure, and manual oligomer modeling utilizing literature information generated a reasonable oligomer model. We also discussed the results of post-prediction docking calculations with AlphaFold2 monomers as input in comparison to our blind prediction results. Overall, ab initio docking of AlphaFold2 models did not lead to better oligomer structure prediction, which may be attributed to the interfacial structural difference between the AlphaFold2 monomer structures and the crystal oligomer structures. This result poses a next-stage challenge in oligomer structure prediction after the success of AlphaFold2. For successful protein assembly structure prediction, a different approach that exploits further evolutionary information on the interface and/or flexible docking taking the interfacial conformational flexibilities of subunit structures into account is needed.

The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.

The heterotrimeric (αβγ) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (β and γ) contain the Cys2-Cys2 motif, which is capable of forming a stable zinc finger structure in the presence of zinc ions. In this work, comparative analysis of the fragments containing Cys2-Cys2 motifs in the aIF2β and aIF2γ structures from different organisms was carried out and their environments in crystals was analyzed. Based on the obtained data, a conclusion was made that the conformation and role of these fragments in the β- and γ-subunits of the aIF2 are different.

Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (β1, β2, and β5). LMP7 (β5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3β2β3 and rα3β2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3β2β3 and hα3β2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3β2β3 subtype compared to rα3β2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3β2β3 and α3β2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3β2β3 nAChRs.

The N-methyl-D-aspartate receptor (NMDAR) in central nerve system is mostly composed of GluN1 and GluN2 subunits. The classical NMDAR has been intensively studied. However, GluN3‑containing NMDAR is much less expressed and have atypical channel properties. Recently, accumulating evidences have revealed two types of GluN3‑containing NMDAR: glutamate-gated GluN1/GluN2/GluN3 NMDAR and glycine-gated GluN1/GluN3 NMDAR. The former may play important roles in regulating synapse maturation and pruning non-used synapses, and its elevated expression at the adult stage may alter synaptic reorganization in some neuropsychiatric disorders. The latter is expressed in the medial habenula and involves in control of aversion. This article reviews the recent progresses on the expression, functional properties of GluN3‑containing atypical NMDARs and the physiological and pathological relevance.