Differential proteomic analysis demonstrates follicle fluid participate immune reaction and protein translation in yak

Background Ovarian follicle fluid (FF) as a microenvironment surrounding oocyte plays critical roles in physio-biochemical processes of follicle development and oocyte maturation. It is hypothesized that proteins in yak FF participate in the physio-biochemical pathways. The primary aims of this study were to find differentially expressed proteins (DEPs) between mature and immature FF, and to elucidating functions of the mature and immature FF in yak. Results The mature and immature FF samples were obtained from three healthy yaks that were nonpregnant, aged from four to five years, and free from any anatomical reproductive disorders. The FF samples were subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ). The FF samples went through correlation analysis, principle component analysis, and expression pattern analysis based on quantification of the identified proteins. Four hundred sixty-three DEPs between mature and immature FF were identified. The DEPs between the mature and immature FF samples underwent gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) analysis. The DEPs highly expressed in the mature FF mainly took parts in the complement and coagulation cascades, defense response, acute-phase response, response to other organism pathways to avoid invasion of exogenous microorganisms. The complement activation pathway contains eight DEPs, namely C2, C5, C6, C7, C9, C4BPA, CFH, and MBL2. The three DEPs, CATHL4, CHGA, and PGLYRP1, take parts in defense response pathway to prevent invasion of exogenetic microorganism. The coagulation cascades pathway involves many coagulation factors, such as F7, F13A1, FGA, FGB, FGG, KLKB1, KNG1, MASP1, SERPINA1, and SERPIND1. While the DEPs highly expressed in the immature FF participated in protein translation, peptide biosynthetic process, DNA conformation change, and DNA geometric change pathways to facilitate follicle development. The translation pathway contains many ribosomal proteins, such as RPL3, RPL5, RPS3, RPS6, and other translation factors, such as EIF3J, EIF4G2, ETF1, MOV10, and NARS. The DNA conformation change and DNA geometric change involve nine DEPs, DDX1, G3BP1, HMGB1, HMGB2, HMGB3, MCM3, MCM5, MCM6, and RUVBL2. Furthermore, the expressed levels of the main DEPs, C2 and SERPIND1, were confirmed by western blot. Conclusions The differential proteomics revealed the up-regulated DEPs in mature FF take parts in immunoreaction to prevent invasion of microorganisms and the up-regulated DEPs in immature FF participate in protein synthesis, which may improve our knowledge of the follicular microenvironment and its biological roles for reproductive processes in yak. The DEPs, C2 and SERPIND1, can be considered as protein markers for mature yak follicle.

Limb Specific Failure of Proliferation and Translation in the Mesenchyme Leads to Skeletal Defects in Diamond Blackfan Anemia

Ribosomopathies are a class of disorders caused by defects in the structure or function of the ribosome and characterized by tissue-specific abnormalities. Diamond Blackfan anemia (DBA) arises from different mutations, predominantly in genes encoding ribosomal proteins (RPs). Apart from the anemia, skeletal defects are among the most common anomalies observed in patients with DBA, but they are virtually restricted to radial ray and other upper limb defects. What leads to these site-specific skeletal defects in DBA remains a mystery. Using a novel mouse model for RP haploinsufficiency, we observed specific, differential defects of the limbs. Using complementary in vitro and in vivo approaches, we demonstrate that reduced WNT signaling and subsequent increased β-catenin degradation in concert with increased expression of p53 contribute to mesenchymal lineage failure. We observed differential defects in the proliferation and differentiation of mesenchymal stem cells (MSCs) from the forelimb versus the hind limbs of the RP haploinsufficient mice that persisted after birth and were partially rescued by allelic reduction of Trp53. These defects are associated with a global decrease in protein translation in RP haploinsufficient MSCs, with the effect more pronounced in cells isolated from the forelimbs. Together these results demonstrate translational differences inherent to the MSC, explaining the site-specific skeletal defects observed in DBA.

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6–RACK1–ERK1/2–FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.

Antibiotics Alter Pocillopora Coral-Symbiodiniaceae-Bacteria Interactions and Cause Microbial Dysbiosis During Heat Stress

Symbioses between eukaryotes and their associated microbial communities are fundamental processes that affect organisms’ ecology and evolution. A unique example of this is reef-building corals that maintain symbiotic associations with dinoflagellate algae (Symbiodiniaceae) and bacteria that affect coral health through various mechanisms. However, little is understood about how coral-associated bacteria communities affect holobiont heat tolerance. In this study, we investigated these interactions in four Pocillopora coral colonies belonging to three cryptic species by subjecting fragments to treatments with antibiotics intended to suppress the normal bacteria community, followed by acute heat stress. Separate treatments with only antibiotics or heat stress were conducted to compare the effects of individual stressors on holobiont transcriptome responses and microbiome shifts. Across all Pocillopora species examined, combined antibiotics and heat stress treatment significantly altered coral-associated bacteria communities and caused major changes in both coral and Cladocopium algal symbiont gene expression. Individually, heat stress impaired Pocillopora protein translation and activated DNA repair processes, while antibiotics treatments caused downregulation of Pocillopora amino acid and inorganic ion transport and metabolism genes and Cladocopium photosynthesis genes. Combined antibiotics-heat stress treatments caused synergistic effects on Pocillopora and Cladocopium gene expression including enhanced expression of oxidative stress response genes, programed cell death pathways and proteolytic enzymes that indicate an exacerbated response to heat stress following bacteria community suppression. Collectively, these results provide further evidence that corals and their Symbiodiniaceae and bacteria communities engage in highly coordinated metabolic interactions that are crucial for coral holobiont health, homeostasis, and heat tolerance.

Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide

One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered DNAJC3 protein levels on the inhibitory activity of CADA. By comparing WT DNAJC3 with a CADA-resistant DNAJC3 mutant, we observed the enhanced sensitivity of human CD4, PTK7 and ERLEC1 for CADA when DNAJC3 was expressed at high levels. Combined treatment of CADA with a proteasome inhibitor resulted in synergistic inhibition of protein translocation and in the rescue of a small preprotein fraction, which presumably corresponds to the CADA affected protein fraction that is stalled at the Sec61 translocon. We demonstrate that DNAJC3 enhances the protein translation of a reporter protein that is expressed downstream of the CADA-stalled substrate, suggesting that DNAJC3 promotes the clearance of the clogged translocon. We propose a model in which a reduced DNAJC3 level by CADA slows down the clearance of CADA-stalled substrates. This results in higher residual translocation into the ER lumen due to the longer dwelling time of the temporarily stalled substrates in the translocon. Thus, by directly reducing DNAJC3 protein levels, CADA attenuates its net down-modulating effect on its substrates.

Alternative RNA splicing in tumour heterogeneity, plasticity and therapy

ABSTRACT Alternative splicing is a process by which a single gene is able to encode multiple different protein isoforms. It is regulated by the inclusion or exclusion of introns and exons that are joined in different patterns prior to protein translation, thus enabling transcriptomic and proteomic diversity. It is now widely accepted that alternative splicing is dysregulated across nearly all cancer types. This widespread dysregulation means that nearly all cellular processes are affected – these include processes synonymous with the hallmarks of cancer – evasion of apoptosis, tissue invasion and metastasis, altered cellular metabolism, genome instability and drug resistance. Emerging evidence indicates that the dysregulation of alternative splicing also promotes a permissive environment for increased tumour heterogeneity and cellular plasticity. These are fundamental regulators of a patient's response to therapy. In this Review, we introduce the mechanisms of alternative splicing and the role of aberrant splicing in cancer, with particular focus on newfound evidence of alternative splicing promoting tumour heterogeneity, cellular plasticity and altered metabolism. We discuss recent in vivo models generated to study alternative splicing and the importance of these for understanding complex tumourigenic processes. Finally, we review the effects of alternative splicing on immune evasion, cell death and genome instability, and how targeting these might enhance therapeutic efficacy.

HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR

Appended to the 5′ end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1–reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5′ untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

The Study on the Clinical Phenotype and Function of HPRT1 Gene

Background: Lesch-Nyhan disease (LND) is a rare x-linked purine metabolic neurogenetic disease caused by enzyme hypoxanthine-guanine phosphoriribosyltransferase(HGprt) deficiency, also known as self-destructive appearance syndrome. A series of manifestations are caused by abnormal purine metabolism. The typical clinical manifestations are hyperuricemia, growth retardation, mental retardation, short stature, dance-like athetosis, aggressive behavior, and compulsive self-harm.. Results: we identified a point mutation c.151C > T (p. Arg51*) in a pedigree. We analyzed the clinical characteristics of children in a family, and obtained the blood of their parents and siblings for second-generation sequencing. At the same time, we also analyzed and compared the expression of HPRT1 gene and predicted the three-dimensional structure of the protein. And we analyzed the clinical manifestations caused by the defect of the HPRT1 genethe mutation led to the termination of transcription at the 51st arginine, resulting in the production of truncated protein, and the relative expression of HPRT1 gene in patients was significantly lower than other family members and 10 normal individuals. Conclusion: this mutation leads to the early termination of protein translation and the formation of a truncated HPRT protein, which affects the function of the protein and generates corresponding clinical manifestations.