Subunits Of Protein Complexes Research Articles

Cytonuclear evolution in fully heterotrophic plants: lifestyles and gene function determine scenarios

BackgroundEvidence shows that full mycoheterotrophs and holoparasites often have reduced plastid genomes with rampant gene loss, elevated substitution rates, and deeply altered to conventional evolution in mitochondrial genomes, but mechanisms of cytonuclear evolution is unknown. Endoparasitic Sapria himalayana and mycoheterotrophic Gastrodia and Platanthera guangdongensis represent different heterotrophic types, providing a basis to illustrate cytonuclear evolution. Here, we focused on nuclear-encoded plastid / mitochondrial (N-pt / mt) -targeting protein complexes, including caseinolytic protease (ClpP), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), oxidative phosphorylation system (OXPHOS), DNA recombination, replication, and repair (DNA-RRR) system, and pentatricopeptide repeat (PPR) proteins, to identify evolutionary drivers for cytonuclear interaction.ResultsThe severity of gene loss of N-pt PPR and pt-RRR genes was positively associated with increased degree of heterotrophy in full mycoheterotrophs and S. himalayana, while N-mt PPR and mt-RRR genes were retained. Substitution rates of organellar and nuclear genes encoding N-pt/mt subunits in protein complexes were evaluated, cytonuclear coevolution was identified in S. himalayana, whereas disproportionate rates of evolution were observed in the OXPHOS complex in full mycoheterotrophs, only slight accelerations in substitution rates were identified in N-mt genes of full mycoheterotrophs.ConclusionsNuclear compensatory evolution was identified in protein complexes encoded by plastid and N-pt genes. Selection shaping codon preferences, functional constraint, mt-RRR gene regulation, and post-transcriptional regulation of PPR genes all facilitate mito-nuclear evolution. Our study enriches our understanding of genomic coevolution scenarios in fully heterotrophic plants.

Analysis of metastasis‑related risk factors and clinical relevance in adult soft‑tissue sarcoma.

Metastasis occurs in nearly 50% of cases of adult soft-tissue sarcoma (ASTS), leading to a dismal prognosis, with a 2-year survival rate of ~30%. Consequently, a prognostic model that incorporates metastatic characteristics may be instrumental in predicting survival time and in crafting optimal personalized therapeutic strategies for patients with ASTS. In the present study, a prognostic prediction model for ASTS was developed by examining genes that are differentially expressed between non-metastatic and metastatic patients in the Gene Expression Omnibus dataset. The prognostic model, which includes five featured genes [actin γ2 (ACTG2), apolipoprotein D, coatomer protein complex subunit γ2 imprinted transcript 1, collagen type VI α6 chain and osteomodulin], was further validated in patients with ASTS from the Cancer Genome Atlas dataset. Based on these five-gene signatures, patients were categorized into high- and low-risk groups. Functional and pathway analyses revealed disparities in stemness, extracellular matrix and cell adhesion-related pathways between the two risk groups, particularly noting the activation of the PI3K-Akt pathway in high-risk cases. Analysis of immune infiltration also revealed variations in immune microenvironment changes between the two risk groups. Immunohistochemical staining substantiated the prognostic significance of these gene signatures in a specific sarcoma subtype. Additionally, wound-healing and Transwell assays demonstrated that inhibition of ACTG2 by shRNAs curbed cell migration and invasion in a sarcoma HOS cell line, underscoring its role in sarcoma metastasis. In conclusion, the present study successfully developed and validated a metastasis-based prognosis prediction model. This model not only reliably forecasts the survival of patients with ASTS, but also may pave the way for further investigation into the processes underlying sarcoma metastasis, ultimately aiding in the design of tailored therapeutic regimens.

Altered assembly paths mitigate interference among paralogous complexes

Protein complexes are fundamental to all cellular processes, so understanding their evolutionary history and assembly processes is important. Gene duplication followed by divergence is considered a primary mechanism for diversifying protein complexes. Nonetheless, to what extent assembly of present-day paralogous complexes has been constrained by their long evolutionary pathways and how cross-complex interference is avoided remain unanswered questions. Subunits of protein complexes are often stabilized upon complex formation, whereas unincorporated subunits are degraded. How such cooperative stability influences protein complex assembly also remains unclear. Here, we demonstrate that subcomplexes determined by cooperative stabilization interactions serve as building blocks for protein complex assembly. We further develop a protein stability-guided method to compare the assembly processes of paralogous complexes in cellulo. Our findings support that oligomeric state and the structural organization of paralogous complexes can be maintained even if their assembly processes are rearranged. Our results indicate that divergent assembly processes by paralogous complexes not only enable the complexes to evolve new functions, but also reinforce their segregation by establishing incompatibility against deleterious hybrid assemblies.

Exploration of the role of EMC3‑AS1 as a potential diagnostic and prognostic indicator in liver cancer.

The aim of the present study was to evaluate the diagnostic and prognostic significance of the long non-coding RNA (lncRNA) endoplasmic reticulum membrane protein complex subunit 3 antisense RNA 1 (EMC3-AS1) in liver cancer, and its impact on the proliferative and invasive capabilities of liver cancer cells. EMC3-AS1 expression in liver cancer was assessed using data from The Cancer Genome Atlas and three Gene Expression Omnibus datasets, and validated in clinical liver cancer samples using reverse transcription-quantitative PCR. The prognostic and diagnostic potentials of this lncRNA were evaluated using Kaplan-Meier and receiver operating characteristic analyses, respectively. The infiltration of immune cells and differential expression of immune checkpoints (ICs) between high- and low-EMC3-AS1 expression groups were investigated. Therapeutic correlation analyses were also undertaken to assess the impact of EMC3-AS1 in the treatment of liver cancer. In addition, in vitro experiments were conducted using small interfering RNA to knock down the expression of EMC3-AS1 in HepG2, Sk-Hep-1 and Huh-7 cells, and evaluate the effect on cell proliferation, colony formation and migration. The results revealed a significant upregulation of EMC3-AS1 expression in liver cancer tissues compared with that in adjacent normal tissues, which was associated with an unfavorable prognosis and demonstrated diagnostic effectiveness for patients with liver cancer. Furthermore, patients with high EMC3-AS1 expression exhibited increased levels of IC markers in comparison with those with low EMC3-AS1 expression. In addition, EMC3-AS1 was indicated to have clinical significance in the prediction of the response to immunotherapy and chemotherapy. Notably, the in vitro experiments demonstrated that the knockdown of EMC3-AS1 significantly hindered cell proliferation, colony formation and migration. Consequently, it was concluded that EMC3-AS1 is upregulated in liver cancer and serves as a prognostic indicator for unfavorable outcomes in patients with liver cancer. Additionally, targeting EMC3-AS1 through knockdown interventions showed potential in mitigating the ability of liver cancer cells to proliferate and migrate, which highlights its dual role as a biomarker and therapeutic target for liver cancer.

Proteome Birthdating Reveals Age-Selectivity of Protein Ubiquitination

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed “proteome birthdating” that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics from a single biological sample encoding multiple labeling time-points. As a first application of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identify a smaller subset of older ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways.

Natural proteome diversity links aneuploidy tolerance to protein turnover

Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy—an imbalance in chromosome copy numbers—is frequent1,2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory3–5. Here we generate a proteomic resource and merge it with genomic1 and transcriptomic6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins—especially subunits of protein complexes—show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes.

Plasma ALS and Gal-3BP differentiate early from advanced liver fibrosis in MASLD patients

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is estimated to affect 30% of the world’s population, and its prevalence is increasing in line with obesity. Liver fibrosis is closely related to mortality, making it the most important clinical parameter for MASLD. It is currently assessed by liver biopsy – an invasive procedure that has some limitations. There is thus an urgent need for a reliable non-invasive means to diagnose earlier MASLD stages.MethodsA discovery study was performed on 158 plasma samples from histologically-characterised MASLD patients using mass spectrometry (MS)-based quantitative proteomics. Differentially abundant proteins were selected for verification by ELISA in the same cohort. They were subsequently validated in an independent MASLD cohort (n = 200).ResultsFrom the 72 proteins differentially abundant between patients with early (F0-2) and advanced fibrosis (F3-4), we selected Insulin-like growth factor-binding protein complex acid labile subunit (ALS) and Galectin-3-binding protein (Gal-3BP) for further study. In our validation cohort, AUROCs with 95% CIs of 0.744 [0.673 – 0.816] and 0.735 [0.661 – 0.81] were obtained for ALS and Gal-3BP, respectively. Combining ALS and Gal-3BP improved the assessment of advanced liver fibrosis, giving an AUROC of 0.796 [0.731. 0.862]. The {ALS; Gal-3BP} model surpassed classic fibrosis panels in predicting advanced liver fibrosis.ConclusionsFurther investigations with complementary cohorts will be needed to confirm the usefulness of ALS and Gal-3BP individually and in combination with other biomarkers for diagnosis of liver fibrosis. With the availability of ELISA assays, these findings could be rapidly clinically translated, providing direct benefits for patients.Graphical

DeepSub: Utilizing Deep Learning for Predicting the Number of Subunits in Homo-Oligomeric Protein Complexes.

The molecular weight (MW) of an enzyme is a critical parameter in enzyme-constrained models (ecModels). It is determined by two factors: the presence of subunits and the abundance of each subunit. Although the number of subunits (NS) can potentially be obtained from UniProt, this information is not readily available for most proteins. In this study, we addressed this gap by extracting and curating subunit information from the UniProt database to establish a robust benchmark dataset. Subsequently, we propose a novel model named DeepSub, which leverages the protein language model and Bi-directional Gated Recurrent Unit (GRU), to predict NS in homo-oligomers solely based on protein sequences. DeepSub demonstrates remarkable accuracy, achieving an accuracy rate as high as 0.967, surpassing the performance of QUEEN. To validate the effectiveness of DeepSub, we performed predictions for protein homo-oligomers that have been reported in the literature but are not documented in the UniProt database. Examples include homoserine dehydrogenase from Corynebacterium glutamicum, Matrilin-4 from Mus musculus and Homo sapiens, and the Multimerins protein family from M. musculus and H. sapiens. The predicted results align closely with the reported findings in the literature, underscoring the reliability and utility of DeepSub.

Development of a New Model System to Study Long-Distance Interactions Supported by Architectural Proteins.

Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer-promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.

Proteome analysis develops novel plasma proteins classifier in predicting the mortality of COVID-19.

COVID-19 has been a global concern for 3 years, however, consecutive plasma protein changes in the disease course are currently unclear. Setting the mortality within 28 days of admission as the main clinical outcome, plasma samples were collected from patients in discovery and independent validation groups at different time points during the disease course. The whole patients were divided into death and survival groups according to their clinical outcomes. Proteomics and pathway/network analyses were used to find the differentially expressed proteins and pathways. Then, we used machine learning to develop a protein classifier which can predict the clinical outcomes of the patients with COVID-19 and help identify the high-risk patients. Finally, a classifier including C-reactive protein, extracellular matrix protein 1, insulin-like growth factor-binding protein complex acid labile subunit, E3 ubiquitin-protein ligase HECW1 and phosphatidylcholine-sterol acyltransferase was determined. The prediction value of the model was verified with an independent patient cohort. This novel model can realize early prediction of 28-day mortality of patients with COVID-19, with the area under curve 0.88 in discovery group and 0.80 in validation group, superior to 4C mortality and E-CURB65 scores. In total, this work revealed a potential protein classifier which can assist in predicting the outcomes of COVID-19 patients and providing new diagnostic directions.

Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome.

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

Endoplasmic Reticulum Membrane Protein Complex Regulates Cancer Stem Cells and is Associated with Sorafenib Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, underscoring the need for novel therapeutic targets. This study aimed to elucidate the role of endoplasmic reticulum membrane protein complex subunit 1 (EMC1) in HCC progression and its therapeutic potential. Publicly available sequencing data and biopsy specimens were analyzed to assess EMC's clinical value and functions in HCC. In vitro experiments validated EMC functions, and multiplex immunofluorescence analysis examined EMC-associated sorafenib resistance mechanisms. EMC1 expression was knocked down in HCC cell lines, followed by cell viability, wound healing, and transwell migration assays. Tumor growth and response to sorafenib treatment were evaluated in mouse models. Metabolomic analysis assessed changes in the TCA cycle. EMC genes were aberrantly expressed in HCC, and high EMC1 expression correlated with poorer survival rates. EMC1 disruption enhanced HCC cells' sensitivity to sorafenib, reducing cell viability, increasing apoptosis, and decreasing tumor size and weight. EMC1 maintained cancer cell stemness and promoted M2 macrophage infiltration. Metabolomic analysis revealed significant changes in the TCA cycle, indicating EMC1's role in HCC metabolic reprogramming. Importantly, EMC1 is highly associated with sorafenib resistance, potentially linked to CTNNB1 mutation or activation. EMC1 plays a critical role in regulating the sorafenib resistance in HCC. Targeting EMC1 may improve HCC treatment efficacy.

Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons

Selective protein degradation typically involves substrate recognition via short linear motifs known as degrons. Various degrons can be found at protein termini from bacteria to mammals. While N-degrons have been extensively studied, our understanding of C-degrons is still limited. Towards a comprehensive understanding of eukaryotic C-degron pathways, here we perform an unbiased survey of C-degrons in budding yeast. We identify over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast C‑terminome. Combining machine learning, high-throughput mutagenesis and genetic screens reveals that the SCF ubiquitin ligase targets ~40% of degrons using a single F-box substrate receptor Das1. Although sequence-specific, Das1 is highly promiscuous, recognizing a variety of C-degron motifs. By screening for full-length substrates, we implicate SCFDas1 in degradation of orphan protein complex subunits. Altogether, this work highlights the variety of C-degron pathways in eukaryotes and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis.

Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli.

Pulmonary host defense is critical for the control of lung infection and inflammation. An increased expression and activity of Toll-like receptor 4 (TLR4) induce phagocytic uptake/clearance and inflammation against Gram-negative bacteria. In this study, we addressed the mechanistic aspect of the immunomodulatory activity of the TLR4-interacting SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) against Escherichia coli. Binding of the SPA4 peptide to bacteria and direct anti-bacterial effects were investigated using flow cytometric, microscopic, and bacteriological methods. The bacterial uptake and inflammatory cytokine response were studied in dendritic cells expressing endogenous basal level of TLR4 or overexpressing TLR4. The subcellular distribution and co-localization of TLR4 and bacteria were investigated by immunocytochemistry. Furthermore, we studied the cellular expression and co-localization of endoplasmic reticulum (ER) molecules (calnexin and ER membrane protein complex subunit 1; EMC1) with lysosomal-associated membrane protein 1 (LAMP1) in cells infected with E. coli and treated with the SPA4 peptide. Simultaneously, the expression of histone H2A protein was quantitated by immunoblotting. Our results demonstrate no binding or direct killing of the bacteria by SPA4 peptide. Instead, it induces the uptake and localization of E. coli in the phagolysosomes for lysis and simultaneously suppresses the secreted levels of TNF-α. Overexpression of TLR4 further augments the pro-phagocytic and anti-inflammatory activity of SPA4 peptide. A time-dependent change in subcellular distribution of TLR4 and an increased co-localization of TLR4 with E. coli in SPA4 peptide-treated cells suggest an enhanced recognition and internalization of bacteria in conjugation with TLR4. Furthermore, an increased co-localization of calnexin and EMC1 with LAMP1 indicates the involvement of ER in pro-phagocytic activity of SPA4 peptide. Simultaneous reduction in secreted amounts of TNF-α coincides with suppressed histone H2A protein expression in the SPA4 peptide-treated cells. These results provide initial insights into the plausible role of ER and histones in the TLR4-immunomodulatory activity of SPA4 peptide against Gram-negative bacteria.

Novel insight into the phenotype of microcephaly 19 in the patient with missense COPB2 mutation

COPB2 gene encodes the Coatomer Protein Complex Subunit Beta-2 that plays a crucial role in the cellular vesicle transport system and it is essential for brain development during embryogenesis. Mutations in COPB2 lead to an extremely rare genetic disease named Microcephaly type 19 with autosomal recessive inheritance. This study describes a missense pathogenic homozygous variant (NM_004766.3:c.760 C > T, p.Arg254Cys) in the COPB2 gene, which was identified by Whole-Exome sequencing and confirmed by Sanger sequencing. The proband of the present study is an eight-and-a-half-year-old Iranian female who was born to consanguineous parents. She manifests global developmental delay, intellectual disability, microcephaly, seizures, spasticity, strabismus, and failure to thrive symptoms. Moreover, she is unable to stand, walk, or speak. Here we report the second homozygous mutation (NM_004766.3:c.760 C > T, p.Arg254Cys) in the COPB2 gene in the second family in the world with MCPH19. The responsible variant (NM_004766.3:c.760 C > T, p.Arg254Cys) for the observed symptoms in the proband was identical to the identified variant in the previously reported Caucasian/Native American family. Sharing this extremely rare pathogenic variant in two families with different origins is an extraordinary event that could aid us to determine the phenotype of this disease more precisely. Eventually, we provide a case-based review of the clinical features and compared our findings to the previously reported family for a better understanding of the clinical presentation of Microcephaly type 19 disease.

Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3

Inherited retinal dystrophies (IRDs) are a heterogeneous group of visual disorders caused by different pathogenic mutations in genes and regulatory sequences. The endoplasmic reticulum (ER) membrane protein complex (EMC) subunit 3 (EMC3) is the core unit of the EMC insertase that integrates the transmembrane peptides into lipid bilayers, and the function of its cytoplasmic carboxyl terminus remains to be elucidated. In this study, an insertional mutation c.768insT in the C-terminal coding region of EMC3 was identified and associated with dominant IRDs in a five-generation family. This mutation caused a frameshift in the coding sequence and a gain of an additional 16 amino acid residues (p.L256F-fs-ext21) to form a helix structure in the C-terminus of the EMC3 protein. The mutation is heterozygous with an incomplete penetrance, and cosegregates in all patients examined. This finding indicates that the C-terminus of EMC3 is essential for EMC functions and that EMC3 may be a novel candidate gene for retinal degenerative diseases.

Novel "GaEl Antigenic Patches" Identified by a "Reverse Epitomics" Approach to Design Multipatch Vaccines against NIPAH Infection, a Silent Threat to Global Human Health.

Nipah virus (NiV) is a zoonotic virus that causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. Several NiV outbreaks have been reported since 1999 with nearly annual occurrences in Bangladesh. The outbreaks had high mortality rates ranging from 40 to 90%. No specific vaccine has yet been reported against NiV. Recently, several vaccine candidates and different designs of vaccines composed of epitopes against NiV were proposed. Most of the vaccines target single protein or protein complex subunits of the pathogen. The multiepitope vaccines proposed also cover a largely limited number of epitopes, and hence, their efficiency is still uncertain. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have utilized the "reverse epitomics" approach ("overlapping-epitope-clusters-to-patches" method) to identify "antigenic patches" (Ag-Patches) and utilize them as immunogenic composition for multipatch vaccine (MPV) design. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties, and interaction with Toll-like receptor 3 ectodomain. In total, 30 CTL (cytotoxic T lymphocyte) and 27 HTL (helper T lymphocyte) antigenic patches were identified from the entire NiV proteome based on the clusters of overlapping epitopes. These identified Ag-Patches cover a total of discrete 362 CTL and 414 HTL epitopes from the entire proteome of NiV. The antigenic patches were utilized as immunogenic composition for the design of two CTL and two HTL multipatch vaccines. The 57 antigenic patches utilized here cover 776 overlapping epitopes targeting 52 different HLA class I and II alleles, providing a global ethnically distributed human population coverage of 99.71%. Such large number of epitope coverage resulting in large human population coverage cannot be reached with single-protein/subunit or multiepitope based vaccines. The reported antigenic patches also provide potential immunogenic composition for early detection diagnostic kits for NiV infection. Further, all the MPVs and Toll-like receptor ectodomain complexes show a stable nature of molecular interaction with numerous hydrogen bonds, salt bridges, and nonbounded contact formation and acceptable root mean square deviation and fluctuation. The cDNA analysis shows a favorable large-scale expression of the MPV constructs in a human cell line. By utilizing the novel "reverse epitomics" approach, highly immunogenic novel "GaEl antigenic patches" (GaEl Ag-Patches), a synonym term for "antigenic patches", were identified and utilized as immunogenic composition to design four MPVs against NiV. We conclude that the novel multipatch vaccines are potential candidates to combat NiV, with greater effectiveness, high specificity, and large human population coverage worldwide.

A previously uncharacterized divisome-associated lipoprotein, DalA, is needed for normal cell division in Rhodobacterales.

The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria. Using Rhodobacter sphaeroides, we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides' divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria. Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.

Strumpellin/WASHC5 regulates the structural plasticity of cortical neurons involved in gait coordination

Strumpellin/Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex subunit 5 (WASHC5) is a core component of the WASH complex, and its mutations confer pathogenicity for hereditary spastic paraplegia (HSP) type SPG8, a rare neurodegenerative gait disorder. WASH complex activates actin-related protein-2/3-mediated actin polymerization and plays a pivotal role in intracellular membrane trafficking in endosomes. In this study, we examined the role of strumpellin in the regulation of structural plasticity of cortical neurons involved in gait coordination. Administration of a lentivirus containing a strumpellin-targeting short hairpin RNA (shRNA) to cortical motor neurons lead to abnormal motor coordination in mice. Strumpellin knockdown using shRNA attenuated dendritic arborization and synapse formation in cultured cortical neurons, and this effect was rescued by wild-type strumpellin expression. Compared with the wild-type, strumpellin mutants N471D or V626F identified in patients with SPG8 exhibited no differences in rescuing the defects. Moreover, the number of F-actin clusters in neuronal dendrites was decreased by strumpellin knockdown and rescued by strumpellin expression. In conclusion, our results indicate that strumpellin regulates the structural plasticity of cortical neurons via actin polymerization.

1631-P: scEMC10 Suppresses Glucose Uptake in Skeletal Muscle via the Inhibition of AMPK Signaling

Skeletal muscle is one of the most important organs involved in the glucose metabolism, and is mainly responsible for postprandial glucose uptake. We have previously shown that overexpression of the secreted isoform of endoplasmic reticulum protein complex subunit 10 (scEMC10) impairs, while Emc10 knockout (KO) or antibody neutralization of circulating scEMC10 improves glucose tolerance in mice. In this study, we presented evidence the regulation of glucose metabolism is achieved through the impacts of scEMC10 on skeletal muscle. PET-CT analyses showed Emc10 KO increased, whereas overexpression of scEMC10 decreased glucose uptake of skeletal muscle in mice, which can be accounted for by the corresponding changes in GLUT4 expression in their skeletal muscle tissues. Recombinant scEMC10 protein reduced both expression and membrane translocation of GLUT4, thus inhibiting glucose uptake in L6-GLUT4myc skeletal muscle cells. Mechanistically, scEMC10 decreased phosphorylations of both AMPK and its downstream target TBC1D1 in both skeletal muscle cells and tissues. Intraperitoneal administration of a scEMC10 neutralizing antibody increased both expression and membrane translocation of GLUT4 accompanied by increased uptake of glucose in gastrocnemius in mice. These data support scEMC10 as an inhibitor of glucose uptake in skeletal muscle and suggest the inhibition of scEMC10 will be a promising way to lower blood glucose in diabetes. Disclosure S.Jin: None. Y.Wang: None. K.Chen: None. J.Dai: None. L.Chen: None. S.Liu: None. X.Wang: None. Funding National Natural Science Foundation of China (81873645); Science and Technology Commission of Shanghai Municipality (22140902700)