Protein Transport Pathways Research Articles

Comparative analysis of testicular fusion in Spodoptera litura (cutworm) and Bombyx mori (silkworm): Histological and transcriptomic insights

Spodoptera litura commonly known as the cutworm, is among the most destructive lepidopteran pests affecting over 120 plants species. The powerful destructive nature of this lepidopteran is attributable to its high reproductive capacity. The testicular fusion that occurs during metamorphosis from larvae to pupa in S.litura positively influences the reproductive success of the offspring. In contrast, Bombyx mori, the silkworm, retains separate testes throughout its life and does not undergo this fusion process. Microscopic examination reveals that during testicular fusion in S.litura, the peritoneal sheath becomes thinner and more translucent, whereas in B.mori, the analogous region thickens. The outer basement membrane in S.litura exhibits fractures, discontinuity, and uneven thickness accompanied by a significant presence of cellular secretions, large cell size, increased vesicles, liquid droplets, and a proliferation of rough endoplasmic reticulum and mitochondria. In contrast, the testicular peritoneal sheath of B.mori at comparable developmental stage exhibits minimal change. Comparative transcriptomic analysis of the testicular peritoneal sheath reveals a substantial difference in gene expression between the two species. The disparity in differential expressed genes (DEGs) is linked to an enrichment of numerous transcription factors, intracellular signaling pathways involving Ca2+ and GTPase, as well as intracellular protein transport and signaling pathways. Meanwhile, structural proteins including actin, chitin-binding proteins, membrane protein fractions, cell adhesion, extracellular matrix proteins are predominantly identified. Moreover, the study highlights the enrichment of endopeptidases, serine proteases, proteolytic enzymes and matrix metalloproteins, which may play a role in the degradation of the outer membrane. Five transcription factors-Slforkhead, Slproline, Slcyclic, Slsilk, and SlD-ETS were identified, and their expression pattern were confirmed by qRT-PCR. they are candidates for participating in the regulation of testicular fusion. Our findings underscore significant morphological and trancriptomic variation in the testicular peritoneal sheath of S.litura compared to the silkworm, with substantial changes at the transcriptomic level coinciding with testicular fusion. The research provides valuable clues for understanding the complex mechanisms underlying this unique phenomenon in insects.

Unraveling the unfolded protein response signature: implications for tumor immune microenvironment heterogeneity and clinical prognosis in stomach cancer.

Stomach cancer is a leading cause of cancer-related deaths globally due to its high grade and poor response to treatment. Understanding the molecular network driving the rapid progression of stomach cancer is crucial for improving patient outcomes. This study aimed to investigate the role of unfolded protein response (UPR) related genes in stomach cancer and their potential as prognostic biomarkers. RNA expression data and clinical follow-up information were obtained from the TCGA and GEO databases. An unsupervised clustering algorithm was used to identify UPR genomic subtypes in stomach cancer. Functional enrichment analysis, immune landscape analysis, and chemotherapy benefit prediction were conducted for each subtype. A prognostic model based on UPR-related genes was developed and validated using LASSO-Cox regression, and a multivariate nomogram was created. Key gene expression analyses in pan-cancer and in vitro experiments were performed to further investigate the role of the identified genes in cancer progression. A total of 375 stomach cancer patients were included in this study. Analysis of 113 UPR-related genes revealed their close functional correlation and significant enrichment in protein modification, transport, and RNA degradation pathways. Unsupervised clustering identified two molecular subtypes with significant differences in prognosis and gene expression profiles. Immune landscape analysis showed that UPR may influence the composition of the tumor immune microenvironment. Chemotherapy sensitivity analysis indicated that patients in the C2 molecular subtype were more responsive to chemotherapy compared to those in the C1 molecular subtype. A prognostic signature consisting of seven UPR-related genes was constructed and validated, and an independent prognostic nomogram was developed. The gene IGFBP1, which had the highest weight coefficient in the prognostic signature, was found to promote the malignant phenotype of stomach cancer cells, suggesting its potential as a therapeutic target. The study developed a UPR-related gene classifier and risk signature for predicting survival in stomach cancer, identifying IGFBP1 as a key factor promoting the disease's malignancy and a potential therapeutic target. IGFBP1's role in enhancing cancer cell adaptation to endoplasmic reticulum stress suggests its importance in stomach cancer prognosis and treatment.

Global genomic diversity and conservation of SARS-CoV-2 since the COVID-19 outbreak.

Global sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has enabled researchers to monitor the genetic information of new pathogenic variants. However, the integrity and accuracy of most genomic information cannot be guaranteed due to limited sample quality and sequencing technology. This restricts researchers from tracking the latest changes in the virus genome. In this study, we aimed to characterize the diversity and conservation balance of complete SARS-CoV-2 genomes worldwide since the outbreak. To achieve this goal, we collected 5,966,490 genome sequences from various parts of the world, excluding those with incomplete or unknown/degenerate sequences. Our methodology included comparing sequences using BLASTN and BLASTP, analyzing RNA secondary structure, and calculating entropy. Our findings provide insights into the characteristics of SARS-CoV-2 and potential mechanisms of pathogenesis. We found that uracil had the highest proportion of all bases among various coronaviruses and cytosine to uracil mutations had the highest proportion among all point mutations. The consistency in the front part (1-26,599 nt) was significantly higher than that in the back part (26,600-29,903 nt) of the genome. For most genes, the similar consistency characteristics were also observed in their protein families. IMPORTANCE Our results indicate that most severe acute respiratory syndrome coronavirus 2 genomes sampled from patients had a mutation rate ≤1.07 ‰ and genome-tail proteins (including S protein) were the main sources of genetic polymorphism. The analysis of the virus-host interaction network of genome-tail proteins showed that they shared some antiviral signaling pathways, especially the intracellular protein transport pathway.

Novel variants in genes related to vesicle-mediated-transport modify Parkinson's disease risk.

VPS35 and VPS13 have been associated with Parkinson's disease (PD), and their shared phenotype in yeast when reduced in function is abnormal vacuolar transport. We aim to test if additional potentially deleterious variants in other genes that share this phenotype can modify the risk for PD. 77 VPS and VPS-related genes were analyzed using whole-genome-sequencing data from 202 PD patients of Ashkenazi Jewish (AJ) ancestry. Filtering was done based on quality and functionality scores. Ten variants in nine genes were further genotyped in 1200 consecutively recruited unrelated AJ-PD patients, and allele frequencies and odds ratio calculated compared to gnomAD-AJ-non-neuro database, in un-stratified (n=1200) and stratified manner (LRRK2-G2019S-PD patients (n=145), GBA-PD patients (n=235), and non-carriers of these mutations (NC, n=787)). Five variants in PIK3C3, VPS11, AP1G2, HGS and VPS13D were significantly associated with PD-risk. PIK3C3-R768W showed a significant association in an un-stratified (all PDs) analysis, as well as in stratified (LRRK2, GBA, and NC) analyses (Odds ratios=2.71, 5.32, 3.26. and 2.19 with p=0.0015, 0.002, 0.0287, and 0.0447, respectively). AP1G2-R563W was significantly associated in LRRK2-carriers (OR=3.69, p=0.006) while VPS13D-D2932N was significantly associated in GBA-carriers (OR=5.45, p=0.0027). VPS11-C846G and HGS-S243Y were significantly associated in NC (OR=2.48 and 2.06, with p=0.022 and 0.0163, respectively). Variants in genes involved in vesicle-mediated protein transport and recycling pathways, including autophagy and mitophagy, may differentially modify PD-risk in LRRK2-carriers, GBA carriers, or NC. Specifically, PIK3C3-R768W is a PD-risk allele, with the highest effect size in LRRK2-G2019S carriers. These results suggest oligogenic effect that may depends on the genetic background of the patient. An unbiased burden of mutations approach in these genes should be evaluated in additional PD and control groups. The mechanisms by which these novel variants interact and increase PD-risk should be researched in depth for better tailoring therapeutic intervention for PD prevention or slowing disease progression.

Regulation of Protein Transport Pathways by the Cytosolic Hsp90s.

The highly conserved molecular chaperone heat shock protein 90 (Hsp90) is well-known for maintaining metastable proteins and mediating various aspects of intracellular protein dynamics. Intriguingly, high-throughput interactome studies suggest that Hsp90 is associated with a variety of other pathways. Here, we will highlight the potential impact of Hsp90 in protein transport. Currently, a limited number of studies have defined a few mechanistic contributions of Hsp90 to protein transport, yet the relevance of hundreds of additional connections between Hsp90 and factors known to aide this process remains unresolved. These interactors broadly support transport pathways including endocytic and exocytic vesicular transport, the transfer of polypeptides across membranes, or unconventional protein secretion. In resolving how Hsp90 contributes to the protein transport process, new therapeutic targets will likely be obtained for the treatment of numerous human health issues, including bacterial infection, cancer metastasis, and neurodegeneration.

In silico identification of miRNAs potentially targeting the genes that regulate protein transport and translocation in cattle in heat stress response

MicroRNAs (miRNAs) are involved in post-transcriptional gene regulation and single miRNAs regulating multiple mRNAs targets. Using bioinformatics approaches, this work aims to identify and characterize the miRNAs that could potentially bind with PDKCC, AP3S2 and SEC61A2 gene which regulate protein transport during heat stress. To resolve this problem, three freely available online miRNAs target finder databases were applied to identify the potential miRNAs which may target PDKCC, AP3S2 and SEC61A2 gene. The prediction results showed that a total of 115, 67 and 25 miRNAs for PDKCC, 267, 111 and 12 miRNAs for AP3S2 and 92, 120 and 31 miRNAs for SEC61A2 were identified as a possible potential candidate by TargetScan, MirTarget2 and miRanda respectively. Following the clustering process, we found eight miRNAs for PDKCC, three for AP3S2 and nine for SEC61A2s identified as possible candidates by all three databases. Five miRNAs in PDKCC, four in AP3S2 and five in the SEC61A2 gene have 100% identical sequence resemblance between human and bovine miRNAs. miRNAs binding site analysis showed that miR-34a in AP3S2 and miR-377 in SEC61A2 has the highest number of binding sites while miR-30b and miR-30c have two binding sites in 3´UTR of SEC61A2 gene whereas the rest of the miRNAs have the lowest number of the binding site in PDKCC and SEC61A2 gene respectively. Our study identified total four potential miRNAs that have different binding sites at the AP3S2 and SEC61A2 3´UTR site along with seven miRNAs which have single binding site and can modulate protein transport, translocation and other cellular pathways.

Gene co-expression architecture in peripheral blood in a cohort of remitted first-episode schizophrenia patients

A better understanding of schizophrenia subtypes is necessary to stratify the patients according to clinical attributes. To explore the genomic architecture of schizophrenia symptomatology, we analyzed blood co-expression modules and their association with clinical data from patients in remission after a first episode of schizophrenia. In total, 91 participants of the 2EPS project were included. Gene expression was assessed using the Clariom S Human Array. Weighted-gene co-expression network analysis (WGCNA) was applied to identify modules of co-expressed genes and to test its correlation with global functioning, clinical symptomatology, and premorbid adjustment. Among the 25 modules identified, six modules were significantly correlated with clinical data. These modules could be clustered in two groups according to their correlation with clinical data. Hub genes in each group showing overlap with risk genes for schizophrenia were enriched in biological processes related to metabolic processes, regulation of gene expression, cellular localization and protein transport, immune processes, and neurotrophin pathways. Our results indicate that modules with significant associations with clinical data showed overlap with gene sets previously identified in differential gene-expression analysis in brain, indicating that peripheral tissues could reveal pathogenic mechanisms. Hub genes involved in these modules revealed multiple signaling pathways previously related to schizophrenia, which may represent the complex interplay in the pathological mechanisms behind the disease. These genes could represent potential targets for the development of peripheral biomarkers underlying illness traits in clinical remission stages after a first episode of schizophrenia.

A human brain vascular atlas reveals diverse mediators of Alzheimer's risk.

The human brain vasculature is of great medical importance: its dysfunction causes disability and death1, and the specialized structure it forms-the blood-brain barrier-impedes the treatment of nearly all brain disorders2,3. Yet so far, we have no molecular map of the human brain vasculature. Here we develop vessel isolation and nuclei extraction for sequencing (VINE-seq) to profile the major vascular and perivascular cell types of the human brain through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 9 individuals with Alzheimer's disease and 8 individuals with no cognitive impairment. We identify brain-region- and species-enriched genes and pathways. We reveal molecular principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In Alzheimer's disease, we observe selective vulnerability of ECM-maintaining pericytes and gene expression patterns that implicate dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 genes that have been linked to Alzheimer's diseaserisk by genome-wide association studies (GWASs) are expressed in the human brain vasculature, and we confirm this by immunostaining. Vascular GWAS genes map to endothelial protein transport, adaptive immune and ECM pathways. Many are microglia-specific in mice, suggesting a partial evolutionary transfer of Alzheimer's disease risk. Our work uncovers the molecular basis of the human brain vasculature, which will inform our understanding of overall brain health, disease and therapy.

Coping with Abiotic Stress in Plants-An Endomembrane Trafficking Perspective.

Plant cells face many changes through their life cycle and develop several mechanisms to cope with adversity. Stress caused by environmental factors is turning out to be more and more relevant as the human population grows and plant cultures start to fail. As eukaryotes, plant cells must coordinate several processes occurring between compartments and combine different pathways for protein transport to several cellular locations. Conventionally, these pathways begin at the ER, or endoplasmic reticulum, move through the Golgi and deliver cargo to the vacuole or to the plasma membrane. However, when under stress, protein trafficking in plants is compromised, usually leading to changes in the endomembrane system that may include protein transport through unconventional routes and alteration of morphology, activity and content of key organelles, as the ER and the vacuole. Such events provide the tools for cells to adapt and overcome the challenges brought on by stress. With this review, we gathered fragmented information on the subject, highlighting how such changes are processed within the endomembrane system and how it responds to an ever-changing environment. Even though the available data on this subject are still sparse, novel information is starting to untangle the complexity and dynamics of protein transport routes and their role in maintaining cell homeostasis under harsh conditions.

PncsHub: a platform for annotating and analyzing non-classically secreted proteins in Gram-positive bacteria.

From industry to food to health, bacteria play an important role in all facets of life. Some of the most important bacteria have been purposely engineered to produce commercial quantities of antibiotics and therapeutics, and non-classical secretion systems are at the forefront of these technologies. Unlike the classical Sec or Tat pathways, non-classically secreted proteins share few common characteristics and use much more diverse secretion pathways for protein transport. Systematically categorizing and investigating the non-classically secreted proteins will enable a deeper understanding of their associated secretion mechanisms and provide a landscape of the Gram-positive secretion pathway distribution. We therefore developed PncsHub (https://pncshub.erc.monash.edu/), the first universal platform for comprehensively annotating and analyzing Gram-positive bacterial non-classically secreted proteins. PncsHub catalogs 4,914 non-classically secreted proteins, which are delicately categorized into 8 subtypes (including the ‘unknown’ subtype) and annotated with data compiled from up to 26 resources and visualisation tools. It incorporates state-of-the-art predictors to identify new and homologous non-classically secreted proteins and includes three analytical modules to visualise the relationships between known and putative non-classically secreted proteins. As such, PncsHub aims to provide integrated services for investigating, predicting and identifying non-classically secreted proteins to promote hypothesis-driven laboratory-based experiments.

Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization

The proper functioning of many proteins requires their transport to the correct cellular compartment or their secretion. Signal recognition particle (SRP) is a major protein transport pathway responsible for the co-translational movement of integral membrane proteins as well as periplasmic proteins. Deinococcus radiodurans is a ubiquitous bacterium that expresses a complex phenotype of extreme oxidative stress resistance, which depends on proteins involved in DNA repair, metabolism, gene regulation, and antioxidant defense. These proteins are located extracellularly or subcellularly, but the molecular mechanism of protein localization in D. radiodurans to manage oxidative stress response remains unexplored. In this study, we characterized the SRP complex in D. radiodurans R1 and showed that the knockdown (KD) of the SRP RNA (Qpr6) reduced bacterial survival under hydrogen peroxide and growth under chronic ionizing radiation. Through LC-mass spectrometry (MS/MS) analysis, we detected 162 proteins in the periplasm of wild-type D. radiodurans, of which the transport of 65 of these proteins to the periplasm was significantly reduced in the Qpr6 KD strain. Through Western blotting, we further demonstrated the localization of the catalases in D. radiodurans, DR_1998 (KatE1) and DR_A0259 (KatE2), in both the cytoplasm and periplasm, respectively, and showed that the accumulation of KatE1 and KatE2 in the periplasm was reduced in the SRP-defective strains. Collectively, this study establishes the importance of the SRP pathway in the survival and the transport of antioxidant proteins in D. radiodurans under oxidative stress.

Sunflower storage proteins are transported in dense vesicles that contain proteins homologous to the pumpkin vacuolar sorting receptor PV 72

Storage proteins are transported to a special storage compartments in seeds by Golgi dependent or independent pathways depending on the plant species. The aim of this work was to study the sunflower storage protein transport pathway and identified component of the sorting machinery. Immature sunflower seeds were analyzed by subcellular fractionation (using percoll and sucrose gradients) and electron microscopy. The vesicles isolated with percoll, have precursors of 11S globulins, α-TIP, δ-TIP, BiP, and two proteins that have homology to the pumpkin vacuolar sorting receptor PV72. Sucrose isolated vesicles have the same composition than percoll ones, except for the lack of BiP and the presence of only one protein that has reactivity with pea VSR BP80. Electronic micrographies of developing seeds show that the formation of electron dense aggregates starts in the endoplasmic reticulum, and that these aggregates are very abundant in the trans-Golgi apparatus, where release of dense vesicles happens. These vesicles contain a homolog of PV72 in their membranes. Storage proteins are also detected in multivesicular bodies whose membranes have reactivity with PV72 serum. All these results indicated that sunflower storage proteins are transported to protein storage vacuoles by a Golgi dependent pathway in a process in which homologous of PV72 are involved.

BDNF secretion from C2C12 cells is enhanced by methionine restriction

Brain derived neurotrophic factor (BDNF) is produced in skeletal muscle as a myokine that plays a role in muscle metabolism. However, how metabolic changes affect skeletal muscle BDNF expression and release remains to be fully understood. Amino acid restrictions such as methionine restriction (MR) are considered as an alternative fasting approach. Here we reported that in C2C12 myotubes, MR enhanced BDNF release, which was measured using ELISA, RT-qPCR, cell immunostaining, and Western blot. Inhibition of protein transport pathway blocked the MR enhanced BDNF release, confirming that MR-induced BDNF release involved classic protein secretory pathway. MR increased l-lactate product in media, suggesting that MR promoted glycolysis. Treatment with 2-deoxy glucose (2-DG) attenuated lactate production as well as BDNF release, suggesting that glycolysis is involved in the enhanced BDNF release induced by MR. Moreover, treatment with l-Lactate, the end-product of glycolysis, enhanced BDNF gene expression and release in control cells in a dose dependent manner, suggesting lactate produced by glycolysis may mediate the enhanced BDNF release by MR. Overall, the results of this study suggest that MR promotes BDNF secretion from C2C12 myotubes at least partially via enhancing glycolysis and lactate production.

Inhibition of Human Adenovirus Replication by the Importin α/β1 Nuclear Import Inhibitor Ivermectin.

Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -β1 (Imp-β1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-β1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/β1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/β1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

Directing traffic: Chaperone-mediated protein transport in malaria parasites.

The ability of eukaryotic parasites from the phylum Apicomplexa to cause devastating diseases is predicated upon their ability to maintain faithful and precise protein trafficking mechanisms. Their parasitic life cycle depends on the trafficking of effector proteins to the infected host cell, transport of proteins to several critical organelles required for survival, as well as transport of parasite and host proteins to the digestive organelles to generate the building blocks for parasite growth. Several recent studies have shed light on the molecular mechanisms parasites utilise to transform the infected host cells, transport proteins to essential metabolic organelles and for biogenesis of organelles required for continuation of their life cycle. Here, we review key pathways of protein transport originating and branching from the endoplasmic reticulum, focusing on the essential roles of chaperones in these processes. Further, we highlight key gaps in our knowledge that prevents us from building a holistic view of protein trafficking in these deadly human pathogens.

Roles of Plant-Specific Inserts in Plant Defense.

Ubiquitously expressed in plants, the plant-specific insert (PSI) of typical plant aspartic proteases (tpAPs) has been associated with plant development, stress response, and defense processes against invading pathogens. Despite sharing high sequence identity, structural studies revealed possible different mechanisms of action among species. The PSI induces signaling pathways of defense hormones in vivo and demonstrates broad-spectrum activity against phytopathogens in vitro. Recent characterization of the PSI-tpAP relationship uncovered novel, nonconventional intracellular protein transport pathways and improved tpAP production yields for industrial applications. In spite of research to date, relatively little is known about the structure-function relationships of PSIs. A comprehensive understanding of their biological roles may benefit plant protection strategies against virulent phytopathogens.

EDEM3 Modulates Plasma Triglyceride Level through Its Regulation of LRP1 Expression.

SummaryHuman genetics studies have uncovered genetic variants that can be used to guide biological research and prioritize molecular targets for therapeutic intervention for complex diseases. We have identified a missense variant (P746S) in EDEM3 associated with lower blood triglyceride (TG) levels in >300,000 individuals. Functional analyses in cell and mouse models show that EDEM3 deficiency strongly increased the uptake of very-low-density lipoprotein and thereby reduced the plasma TG level, as a result of up-regulated expression of LRP1 receptor. We demonstrate that EDEM3 deletion up-regulated the pathways for RNA and endoplasmic reticulum protein processing and transport, and consequently increased the cell surface mannose-containing glycoproteins, including LRP1. Metabolomics analyses reveal a cellular TG accumulation under EDEM3 deficiency, a profile consistent with individuals carrying EDEM3 P746S. Our study identifies EDEM3 as a regulator of blood TG, and targeted inhibition of EDEM3 may provide a complementary approach for lowering elevated blood TG concentrations.

Detection and in vitro studies of Cucurbita maxima phloem serpin-1 RNA-binding properties

Apart from being a conduit for photoassimilate transport in plants, the phloem serves as a pathway for transport of proteins and RNAs from sites of their synthesis to distant plant parts. As demonstrated for mRNAs and small RNAs such as miRNA and siRNA, their phloem transport is largely involved in responses to environmental cues including stresses and pathogen attacks. RNA molecules are believed to be transported in the phloem in the form of complexes with RNA-binding proteins; however, proteins forming such complexes are generally poorly studied. Here, we demonstrate that the Cucurbita maxima phloem serpin-1 (CmPS1), which has been previously described as a functional protease inhibitor capable of long-distance transport via the phloem, is able to bind RNA in vitro. Among different RNAs tested, CmPS1 exhibits a preference for imperfect RNA duplexes and the highest affinity to tRNA. A characteristic complex formed by CmPS1 with tRNA is not observed upon CmPS1 binding to tRNA-like structures of plant viruses. Mutational analysis demonstrates that the CmPS1 N-terminal region is not involved in RNA binding. Since antithrombin-III, the human protease inhibitor of serpin family most closely sequence-related to CmPS1, is found to be unable to bind RNA, one can suggest that, in its evolution, CmPS1 has gained the RNA binding capability as an additional function likely relevant to its specific activities in the plant phloem.

Profiling the Cell Surface Landscape of Proteasome Inhibitor-Treated and -Resistant Multiple Myeloma to Guide Immunotherapy Targeting, Diagnosis, and Biology

Introduction: Proteasome inhibitor (PI) resistance remains a major clinical challenge in multiple myeloma (MM). As the proteasome plays a central role in cellular protein homeostasis, we hypothesized both PI treatment and resistance might rewire protein transport and recycling pathways, thereby leading to broad changes in cell surface protein expression. Defining the cell surface proteome has become increasingly important in MM to quantify immunotherapy target expression, identify potential biomarkers of drug response or resistance, and reveal proteins mediating interaction with the tumor microenvironment. Unbiased mass spectrometry approaches allow for profiling of hundreds of surface proteins simultaneously, allowing for novel protein discovery and extending beyond the limits of flow or mass cytometry. Methods: PI-resistant cells derived from cell lines AMO-1, L363, and RPMI-8226, and ARH-77 were grown in 90nM Bortezomib or Carfilzomib as previously described (Soriano et al, Leukemia (2016)). N-glycoproteomics was performed in triplicate on PI-resistant and parental lines. Briefly, glycosylated cell surface proteins were biotinylated prior to enrichment with Neutravidin beads and downstream LC-MS analysis. For perturbation studies, AMO-1 and RPMI-8226 cells were treated for 48 hours with Bortezomib, Lenalidomide, or CB-5083 prior to N-glycoproteomics. Flow cytometry was performed to validate surface protein abundance changes in PI-resistant lines, as well as with MM cell lines and ex vivo MM patient bone marrow mononuclear cells (PBMCs) treated with bortezomib and lenalidomide. For ex vivo MM patient cell surface proteomics, plasma cells were isolated from PBMCs with anti-CD138 magnetic beads prior to N-glycoproteomic analysis. Mass spectrometry data was analyzed in Maxquant and statistical analysis was performed in Perseus and R. Results: Supporting our hypothesis, we found that the MM surfaceome is broadly re-wired in the PI-resistant state. Using N-glycoproteomics, we identified EVI2B, CD53, CD50, and ITGB7 as downregulated and CD151, CD10, and SERC3 as upregulated in PI-resistant myeloma cell lines (up and downregulated defined as at least a +/- log2 fold change of 1.5 and p < .05). Notably, ITGB7 is under development as an immunotherapy target for MM (Hosen et al, Nat Med (2017)). To understand how surface remodeling in the PI-resistant state compares with short-term exposure to PI, we treated MM cells with bortezomib and again found broad surfaceome remodeling, including loss of immunotherapy target BCMA. We identified GITR, CD53, and ITGB7, among other proteins, as downregulated in both bortezomib-exposed and PI-resistant MM, suggesting that expression of proteins involved in therapeutic resistance might be rapidly modulated in initial cycles of therapy. To compare surfaceome changes identified in PI-exposure and resistance and those seen under anti-MM therapeutics with alternative mechanisms of action, we performed N-glycoproteomics on MM cells treated with the clinical immunomodulating agent Lenalidomide and the cytotoxic p97/VCP inhibitor CB-5083. Interestingly, we found MUC1, a proposed myeloma oncoprotein, to be upregulated under lenalidomide in AMO-1 but downregulated in the PI-resistant state. We validated surface protein abundance changes in PI-resistant MM cell lines and drug-treated MM cell lines or ex-vivo MM patient samples by flow cytometry. For additional validation, we are currently optimizing methods for N-glycoproteomics on CD138+ cells isolated from primary MM patient bone marrow aspirates. Initial results suggest several markers identified in cell lines are also highly expressed in primary PI-resistant MM. Conclusions: Unbiased N-glycoproteomics reveals broad cell surface protein remodeling in PI-resistant MM. Short-term treatment with bortezomib or lenalidomide also leads to significant surface alterations, with differential expression of proteins including MUC1, GITR, and BCMA potentially playing a role in drug resistance and informing rational combination with immunotherapies. Disclosures Martin: Roche and Juno: Consultancy; Amgen, Sanofi, Seattle Genetics: Research Funding. Wong:Celgene: Research Funding; Janssen: Research Funding; Roche: Research Funding; Fortis: Research Funding. Wiita:Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; UCSF: Patents & Royalties.

Molecular Mimicry of SecA and Signal Recognition Particle Binding to the Bacterial Ribosome

Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome.IMPORTANCE Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time.