Protein Export Pathway Research Articles

Identification of function modules in the co-expression protein–protein interaction network of Bombyx mori in response to Beauveria bassiana infection

Beauveria bassiana (B. bassiana) is a common fungal disease in sericulture. Previous research has primarily focused on investigating genes involved in innate immunity. However, the response of Bombyx mori (B. mori) to B. bassiana requires the coordination of other biological processes in addition to the immune system. We measured protein expression profile of B. mori after inoculating B. bassiana using iTRAQ technology in previous. Here we constructed a co-expression protein–protein interaction network of B. mori in response to B. bassiana infection. Subnetworks and modules were analyzed, and the functions of these modules were annotated. The results revealed the identification of numerous proteins associated with cellular immunity, including those involved in phagosomes, lysosomes, mTOR signaling, sugar metabolism, and the ubiquitin–proteasome pathway. Meanwhile, we observed that the pathways involved in protein synthesis were activated, including pyruvate and purine metabolism, RNA transport, ribosome, protein processing in endoplasmic reticulum, and protein export pathways, during B. bassiana infection. Based on this analysis, we selected six candidate genes (shock protein, ribosome, translocon, actin muscle-type A2, peptidoglycan recognition protein, and collagenase) that were found to be related to the response to B. bassiana. Further verification experiments demonstrated significant changes in their expression levels after inoculation with B. bassiana. These research findings provide new insights into the molecular mechanism of insect immune response to fungal infection.

Integrated transcriptome and microRNA analysis reveals molecular responses to high-temperature stress in the liver of American shad (Alosa sapidissima)

BackgroundFish reproduction, development and growth are directly affected by temperature, investigating the regulatory mechanisms behind high temperature stress is helpful to construct a finer molecular network. In this study, we systematically analyzed the transcriptome and miRNA information of American shad (Alosa sapidissima) liver tissues at different cultivation temperatures of 24 ℃ (Low), 27 ℃ (Mid) and 30 ℃ (High) based on a high-throughput sequencing platform.ResultsThe results showed that there were 1594 differentially expressed genes (DEGs) and 660 differentially expressed miRNAs (DEMs) in the LowLi vs. MidLi comparison group, 473 DEGs and 84 DEMs in the MidLi vs. HighLi group, 914 DEGs and 442 DEMs in the LowLi vs. HighLi group. These included some important genes and miRNAs such as calr, hsp90b1, hsp70, ssa-miR-125a-3p, ssa-miR-92b-5p, dre-miR-15a-3p and novel-m1018-5p. The DEGs were mainly enriched in the protein folding, processing and export pathways of the endoplasmic reticulum; the target genes of the DEMs were mainly enriched in the focal adhesion pathway. Furthermore, the association analysis revealed that the key genes were mainly enriched in the metabolic pathway. Interestingly, we found a significant increase in the number of genes and miRNAs involved in the regulation of heat stress during the temperature change from 24 °C to 27 °C. In addition, we examined the tissue expression characteristics of some key genes and miRNAs by qPCR, and found that calr, hsp90b1 and dre-miR-125b-2-3p were significantly highly expressed in the liver at 27 ℃, while novel-m0481-5p, ssa-miR-125a-3p, ssa-miR-92b-5p, dre-miR-15a-3p and novel-m1018-5p had the highest expression in the heart at 30℃. Finally, the quantitative expression trends of 10 randomly selected DEGs and 10 DEMs were consistent with the sequencing data, indicating the reliability of the results.ConclusionsIn summary, this study provides some fundamental data for subsequent in-depth research into the molecular regulatory mechanisms of A. sapidissima response to heat stress, and for the selective breeding of high temperature tolerant varieties.

Staphylococcus aureus foldase PrsA contributes to the folding and secretion of protein A

BackgroundStaphylococcus aureus secretes a variety of proteins including virulence factors that cause diseases. PrsA, encoded by many Gram-positive bacteria, is a membrane-anchored lipoprotein that functions as a foldase to assist in post-translocational folding and helps maintain the stability of secreted proteins. Our earlier proteomic studies found that PrsA is required for the secretion of protein A, an immunoglobulin-binding protein that contributes to host immune evasion. This study aims to investigate how PrsA influences protein A secretion.ResultsWe found that in comparison with the parental strain HG001, the prsA-deletion mutant HG001ΔprsA secreted less protein A. Deleting prsA also decreased the stability of exported protein A. Pulldown assays indicated that PrsA interacts with protein A in vivo. The domains in PrsA that interact with protein A are mapped to both the N- and C-terminal regions (NC domains). Additionally, the NC domains are essential for promoting PrsA dimerization. Furthermore, an immunoglobulin-binding assay revealed that, compared to the parental strain HG001, fewer immunoglobulins bound to the surface of the mutant strain HG001ΔprsA.ConclusionsThis study demonstrates that PrsA is critical for the folding and secretion of protein A. The information derived from this study provides a better understanding of virulent protein export pathways that are crucial to the pathogenicity of S. aureus.

Cardiac Transcriptome and Histology of the Heart of the Male Chinese Mitten Crab (Eriocheir sinensis) under High-Temperature Stress

High temperatures are important environmental stressors affecting the metabolism, growth, immunity, and mortality of Chinese mitten crabs (Eriocheir sinensis). In this study, Chinese mitten crabs were divided into two groups and exposed to temperatures of 35 °C (thermal stress group) or 25 °C (control group) for 24 h, and the transcriptome of the heart was analyzed. There were 4007 differentially expressed genes (DEGs) between the thermal stress and the control groups, including 2660 upregulated and 1347 downregulated genes. Heat shock proteins (HSPs) and transcription factors (TFs) were temperature-sensitive DEGs in Chinese mitten crabs. DEGs mainly focused on protein processing in the endoplasmic reticulum, ribosome biogenesis, glycine, serine, and threonine metabolism, protein export, and insect hormone biosynthesis pathways. A total of 28,916 SSRs and 59 TF families, including 851 TFs, were detected among all unigenes of E. sinensis transcripts. The qRT-PCR results for the HSPs and apoptotic DEGs from the heart exhibited the same trends as those in the E. sinensis transcriptome data. Results of light microscopy analyzing histological sections of the heart indicated that most myocardial fibers were lysed, and the number of nuclei and the connective tissue contents between the myocardial layers were both reduced following 35 °C exposure for 24 h.

Avian pathogenic Escherichia coli infection causes infiltration of heterophilic granulocytes of chick tracheal by the complement and coagulation cascades pathway

BackgroundAvian pathogenic Escherichia coli (APEC) causes tracheal damage and heterophilic granulocytic infiltration and inflammation in infected chicks. In this study, we infected chick tracheal tissue with strain AE17 and produced pathological sections with proteomic sequencing. We compared the results of pathological sections from the APEC-infected group with those from the PBS control group; the pathological sections from the experimental group showed hemorrhage, fibrinization, and infiltration of heterophilic granulocytes in the tracheal tissue. In order to explore the effect on proteomics on inflammation and to further search for the caus.ResultsThe tandem mass tag-based (TMT) sequencing analysis showed 224 upregulated and 140 downregulated proteins after infection with the AE17 strain. Based on the results of KEGG in Complement and coagulation cascades, differential protein expression in the Protein export pathway was upregulated.ConclusionsWith these results, we found that chemokines produced by the Complement and coagulation cascades pathway may cause infiltration of heterophilic granulocytes involved in inflammation, as well as antimicrobial factors produced by the complement system to fight the infection together.These results suggest that APEC causes the infiltration of heterophilic granulocytes through the involvement of the complement system with serine protease inhibitors.

Metabolic exploration of the developmental abnormalities and neurotoxicity of Esculentoside B, the main toxic factor in Phytolaccae radix.

P: radix is a perennial herb, and its extracts have various biological properties that make it a potential candidate for the treatment of tumors, edema, and lymphatic stasis. However, the main factor contributing to its toxicity are not clear. Here, we used a zebrafish toxicological model to study the main toxicity factor of P. radix and explore the potential mechanisms involved. The results revealed that Esculentoside B was the major toxic factor of P. radix. Exposure of zebrafish larvae to Esculentoside B caused developmental abnormalities, neurotoxicity and altered locomotor behavior. The combination of AChE activity and the expression levels of genes relevant to CNS development demonstrated that Esculentoside B is neurotoxic to zebrafish larvae, impairs their CNS development, and that AChE may be a toxic target of Esculentoside B. Metabolomic analysis has revealed that Esculentoside B exposure can disrupt D-Amino acid metabolism, protein export, autophagy, and mTOR signaling pathways in zebrafish larvae. These findings provide insights into the molecular mechanisms underlying EsB-induced neurotoxicity in zebrafish, which can facilitate further research and development of P. radix for safe consumption.

Selenium-mediated Cr(VI) reduction and SeNPs synthesis accelerated Bacillus cereus SES to remediate Cr contamination

Microbial biotransformation of Cr(VI) is a sustainable approach to reduce Cr(VI) toxicity and remediate Cr(VI) contamination. In this study, Bacillus cereus SES with the capability of reducing both Cr(VI) and Se(IV) was isolated, and the effect of Se supplementation on Cr(VI) reduction by Bacillus cereus SES was investigated. Se(IV) addition enabled 2.6-fold faster Cr(VI) reduction, while B. cereus SES reduced 96.96% Se(IV) and produced more selenium nanoparticles (SeNPs) in the presence of Cr(VI). Co-reduction products of B. cereus SES on Cr(VI) and Se(IV) were SeNPs adsorbed with Cr(III). The relevant mechanisms were further revealed by proteomics. Se(IV) supplementation mediated the synthesis of Cr(VI) reductants and stress-resistant substances, thus enhancing Cr(VI) resistance and promoting Cr(VI) reduction. Meanwhile, high Se(IV) reduction rate was associated with Cr(VI)-induced electron transport processes, and Cr(VI) mediated the up-regulation of flagellar assembly, protein export and ABC transporters pathways to synthesis and export more SeNPs. Furthermore, Se combined with B. cereus SES had the potential to reduce the toxicity of Cr(VI) via reducing the bioavailability of Cr and improving the bioavailability of Se in soil. Results suggested that Se could be an efficient strategy to enhance the remediation of B. cereus SES on Cr contamination.

Transcriptional response of endometrial cells to Insulin, cultured using microfluidics.

Obesity is a rapidly growing public health issue among women of reproductive age associated with decreased reproductive function including implantation failure. This can result from a myriad of factors including impaired gametes and endometrial dysfunction. The mechanisms of how obesity-related hyperinsulinaemia disrupts endometrial function are poorly understood. We investigated potential mechanisms by which insulin alters endometrial transcript expression. Ishikawa cells were seeded into a microfluidics device attached to a syringe pump to deliver a constant flow rate of 1uL/min of the following: 1) control 2) vehicle control (acetic acid) or, 3) Insulin (10 ng/ml) for 24 hours (n=3 biological replicates). Insulin-induced transcriptomic response of endometrial epithelial cells was determined via RNA sequencing, and DAVID and Webgestalt to identify Gene Ontology (GO) terms and signalling pathways. A Total of 29 transcripts showed differential expression levels across two comparison groups (control v vehicle control; vehicle control v insulin). Nine transcripts were differentially expressed in vehicle control v insulin comparison (p<0.05). Functional annotation analysis of transcripts altered by insulin (n=9) identified three significantly enriched GO terms: SRP-dependent cotranslational protein targeting to membrane, poly(A) binding, and RNA binding (p<0.05). Over-representation analysis found three significantly enriched signalling pathways relating to insulin-induced transcriptomic response: protein export, glutathione metabolism, and ribosome pathways (p<0.05). Transfection of siRNA for RASPN successfully knocked down expression (p<0.05) but this did not have any effect on cellular morphology. Insulin-induced dysregulation of biological functions and pathways highlight potential mechanisms by which high insulin concentrations within maternal circulation may perturb endometrial receptivity.

Integrated analyzes identify CCT3 as a modulator to shape immunosuppressive tumor microenvironment in lung adenocarcinoma

BackgroundChaperonin-containing tailless complex polypeptide 1 (TCP1) subunit 3 (CCT3) has tumor-promoting effects in lung adenocarcinoma (LUAD). This study aims to investigate the molecular mechanisms of CCT3 in LUAD oncogenesis.MethodsThe UALCAN databases, Human Protein Atlas (HPA) and The Cancer Genome Atlas (TCGA) data were used to analyze CCT3 expression in LUAD. Both the Wilcoxon rank-sum test and the regression model were used to investigate the connection between clinicopathologic characteristics of LUAD patients and CCT3 expression. The prognostic value of CCT3 was determined by Cox regression models, the Kaplan-Meier method and Nomogram prediction. Next, we identified the most related genes with CCT3 via GeneMANIA and String databases, and the association between CCT3 and infiltrated immune cells using single-sample Gene Set Enrichment Analysis (ssGSEA). CCT3-related pathway enrichment analysis was investigated by GSEA. Finally, CCT3 roles in cell proliferation and apoptosis of LUAD A549 cells was verified by siRNA (small interfering RNA) mediated CCT3 knockdown.ResultsCCT3 was upregulated in LUAD both in mRNA and protein levels. CCT3 overexpression was associated with clinicopathological characteristics including sex, smoking, T- and N-categories, pathological staging, and a poor prognosis of LUAD patients. GeneMANIA and String databases found a set of CCT3-related genes that are connected to the assembly and stability of proteins involved in proteostasis of cytoskeletal filaments, DNA repair and protein methylation. Furthermore, CCT3 was found to be positively correlated with the infiltrating Th2 cells (r = 0.442, p < 0.01) while negatively correlated with mast cells (r = -0.49, p < 0.01) and immature dendritic cells (iDCs, r = -0.401, p < 0.001) according to ssGSEA analyzes. The pathway analysis based on GSEA method showed that the cell cycle pathway, the protein export pathway, the proteasome pathway and the ribosome pathway are enriched in CCT3 high group, whereas the JAK/STAT pathway, B cell receptor pathway, T cell receptor pathway and toll like receptor pathway were enriched in CCT3 low group. Finally, CCT3 knockdown substantially inhibited proliferation while promoted apoptosis of A549 cells.ConclusionIntegrated analyzes identify CCT3 as a modulator to shape immunosuppressive tumor microenvironment in LUAD and therefore, a prognostic factor for LUAD.

Increased expression of CEP72 predicts poor prognosis in multiple myeloma.

Multiple myeloma (MM) is a fatal hematological malignancy and does not have adequate prognostic indicators. Previous studies indicate that CEP72 is closely related to tumorigenesis and tumor progression. However, the expression and function of CEP72 in multiple myeloma have yet to be elucidated. In this study, we explored the correlation between CEP72 expression and clinicopathological characteristics as well as the impacts of CEP72 expression on the survival of MM patients. In addition, PPI, GSEA and Chemotherapy drug resistance analysis identified the possible mechanism. CEP72 is overexpressed in both MM patients and MM cell lines. Clinically, patients in the CEP72high subgroup were significantly older than those in the CEP72low subgroup (p=0.003). Up-regulation of CEP72 was related to poor overall survival and event-free survival. PPI network showed that CEP72 was related to PCM1, KIZ, OFD1, etc. GSEA analysis showed that CEP72 was enriched in cell cycle, oocyte meiosis, protein export, lysosome and N-glycan biosynthesis pathways. Drug resistance analysis indicated that there was a positive correlation between the CEP72 expression and the IC50 values of 6-mercaptopurine, 8-chloro-adenosine, clofarabine, fludarabine and allopurinol. High CEP72 expression was a poor prognostic factor in patients diagnosed with multiple myeloma.

INTEGRATED BIOINFORMATIC ANALYSIS TO EVALUATE TARGET GENES AND PATHWAYS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Objective: The most common type of leukemia, chronic lymphocytic leukemia (CLL), is characterized by progressive accumulation of monoclonal B cells with a specific immunophenotype in the blood, bone marrow, and lymphoid organ. The goal of this research was to use bioinformatic analysis to comprehend the molecular mechanisms causing CLL and to investigate potential targets for the diagnosis and therapy of CLL.&#x0D; Material and Method: Expression data from CLL patients with accession numbers GSE22529 and GSE26725 were downloaded from the GEO database for bioinformatic analysis. GSE22529 data was studied with samples from 41 CLL patients and 11 healthy groups, while GSE26725 data was studied with blood samples from 12 CLL patients and 5 healthy groups. GEO2R was used to find differentially expressed genes (DEGs) in CLL patient samples and healthy control samples. The DAVID program was used to perform GO and KEGG enrichment analyses on DEGs. Using the Cytoscape software, a protein-protein interaction (PPI) network was created, and hub genes associated with CLL were identified.&#x0D; Result and Discussion: DEGs with p 0.05 and log2FC 0, log2FC&gt;0 were chosen after analysis with GEO2R. In the GSE22529 dataset, 942 genes had higher expression levels in CLL patients compared with controls, while the expression of 1007 genes decreased. In the GSE26725 dataset, CLL patients had lower expression levels for 916 genes compared with controls, while 939 genes showed an increase in expression. 229 DEGs with higher expression levels and 308 DEGs with lower expression levels were found in both sets of data. It has been observed that these common genes, whose expression has changed, are enriched in protein processing in the ER, Chemokine, B-cell receptor, T-cell receptor, protein export pathways. Additionally, DDOST, RPL18, RPL18A, RPL19, RPL31, GNB3, GNB4, GNG11, GNGT1, NEDD8, UBE2M RBX1, FBXO21, SKP1, KLHL9 and CAND1 were identified as the most important genes. Our study's findings demonstrated that newly discovered genes and pathways may be candidates for CLL biomarkers that can be used for both the diagnosis and drug treatment of the disease.

Rescue of Mitochondrial Function in Hutchinson-Gilford Progeria Syndrome by the Pharmacological Modulation of Exportin CRM1.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder caused by the expression of progerin, a mutant variant of Lamin A. Recently, HGPS studies have gained relevance because unraveling its underlying mechanism would help to understand physiological aging. We previously reported that the CRM1-mediated nuclear protein export pathway is exacerbated in HGPS cells, provoking the mislocalization of numerous protein targets of CRM1. We showed that normalization of this mechanism by pharmacologically inhibiting CRM1 with LMB (specific CRM1 inhibitor), mitigates the senescent phenotype of HGPS cells. Since mitochondrial dysfunction is a hallmark of HGPS, in this study we analyze the effect of LMB on mitochondrial function. Remarkably, LMB treatment induced the recovery of mitochondrial function in HGPS cells, as shown by the improvement in mitochondrial morphology, mitochondrial membrane potential, and ATP levels, which consequently impeded the accumulation of ROS but not mitochondrial superoxide. We provide evidence that the beneficial effect of LMB is mechanistically based on a combinatory effect on mitochondrial biogenesis via upregulation of PGC-1α expression (master transcription cofactor of mitochondrial genes), and mitophagy through the recovery of lysosomal content. The use of exportin CRM1 inhibitors constitutes a promising strategy to treat HGPS and other diseases characterized by mitochondrial impairment.

Hcp of the Type VI Secretion System (T6SS) in Acidovorax citrulli Group II Strain Aac5 Has a Dual Role as a Core Structural Protein and an Effector Protein in Colonization, Growth Ability, Competition, Biofilm Formation, and Ferric Iron Absorption.

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3+ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.

The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction

The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.

Ribosome profiling reveals multiple roles of SecA in cotranslational protein export

SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also binds ribosomes, but the role of SecA’s ribosome interaction has been unclear. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA. Our data reveal the widespread accumulation of large periplasmic loops of inner membrane proteins in the cytoplasm during their cotranslational translocation, which are specifically recognized and resolved by SecA in coordination with the proton motive force (PMF). Furthermore, SecA associates with 25% of secretory proteins with highly hydrophobic signal sequences at an early stage of translation and mediates their cotranslational transport. In contrast, the chaperone trigger factor (TF) delays SecA engagement on secretory proteins with weakly hydrophobic signal sequences, thus enforcing a posttranslational mode of their translocation. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria.

Comparison of the Agronomic, Cytological, Grain Protein Characteristics, as Well as Transcriptomic Profile of Two Wheat Lines Derived From Wild Emmer

Two advanced wheat lines BAd7-209 and BAd23-1 without the functional gene GPC-B1 were obtained from a cross between common wheat cultivar Chuannong 16 (CN16) and wild emmer wheat accession D97 (D97). BAd7-209 showed superior quality parameters than those of BAd23-1 and CN16. We found that the components of glutenins and gliadins in BAd7-209 and BAd23-1 were similar, whereas BAd7-209 had higher amount of glutenins and gliadins than those of BAd23-1. RNA sequencing analysis on developing grains of BAd7-209 and BAd23-1 as well as their parents revealed 382 differentially expressed genes (DEGs) between the high–grain protein content (GPC) (D97 + BAd7-209) and the low-GPC (CN16 + BAd23-1) groups. DEGs were mainly associated with transcriptional regulation of the storage protein genes, protein processing in endoplasmic reticulum, and protein export pathways. The upregulated gluten genes and transcription factors (e.g., NAC, MYB, and bZIP) may contribute to the high GPC in BAd7-209. Our results provide insights into the potential regulation pathways underlying wheat grain protein accumulation and contribute to make use of wild emmer for wheat quality improvement.

Tumor microenvironment-related multigene prognostic prediction model for breast cancer.

Background: Breast cancer is an invasive disease with complex molecular mechanisms. Prognosis-related biomarkers are still urgently needed to predict outcomes of breast cancer patients.Methods: Original data were download from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). The analyses were performed using perl-5.32 and R-x64-4.1.1.Results: In this study, 1086 differentially expressed genes (DEGs) were identified in the TCGA cohort; 523 shared DEGs were identified in the TCGA and GSE10886 cohorts. Eight subtypes were estimated using non-negative matrix factorization clustering with significant differences seen in overall survival (OS) and progression-free survival (PFS) (P < 0.01). Univariate Cox analysis and least absolute shrinkage and selection operator (LASSO) regression analysis were performed to develop a related risk score related to the 17 DEGs; this score separated breast cancer into low- and high-risk groups with significant differences in survival (P < 0.01) and showed powerful effectiveness (TCGA all group: 1-year area under the curve [AUC] = 0.729, 3-year AUC = 0.778, 5-year AUC = 0.781). A nomogram prediction model was constructed using non-negative matrix factorization clustering, the risk score, and clinical characteristics. Our model was confirmed to be related with tumor microenvironment. Furthermore, DEGs in high-risk breast cancer were enriched in histidine metabolism (normalized enrichment score [NES] = 1.49, P < 0.05), protein export (NES = 1.58, P < 0.05), and steroid hormone biosynthesis signaling pathways (NES = 1.56, P < 0.05).Conclusions: We established a comprehensive model that can predict prognosis and guide treatment.

Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers

Spider dragline silk is a remarkable fiber made of unique proteins—spidroins—secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (>65.8%) and titer (554.7 mg L–1) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m−3. The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials.

Cadmium induced oxidative stress, endoplasmic reticulum (ER) stress and apoptosis with compensative responses towards the up-regulation of ribosome, protein processing in the ER, and protein export pathways in the liver of zebrafish.

The present study identified that exposure to 5, 10, and 20µg/L Cd for 48 days reduced growth, increased Cd accumulation and levels of reactive oxygen species (ROS) and lipid peroxidation, and induced ER stress and cellular apoptosis in the liver in a dose-dependent manner. However, the survival rate was not affected by Cd. The increased production of ROS might result from reduced catalase (CAT) and copper/zinc-superoxide dismutase (Cu/Zn-SOD) activities, which might trigger ER stress pathways and subsequently induce apoptotic responses, ultimately leading to growth inhibition. Transcriptomic analyses indicated that the differentially expressed genes (DEGs) involved in metabolic pathways were significantly enriched and dysregulated by Cd, suggesting that metabolic disturbances may contribute to Cd toxicity. However, there were increases in glutathione peroxidase (GPX) activity, protein levels of metallothioneins (MTs) and heat shock protein 70 (HSP70), and mRNA levels of sod1, cat, gpx, mt2, and hsp70. Furthermore, DEGs related to ribosome, protein processing in the ER, and protein export pathways were significantly enriched and up-regulated by Cd. These increases may be compensatory responses following oxidative stress, ER stress, and apoptosis to resist negative effects. Taken together, we demonstrated that environmentally relevant levels of Cd induced adaptive responses with compensatory mechanisms in fish, which may help to maintain fish survival at the cost of growth.

Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of Listeria monocytogenes

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SignificanceL. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.