Showing papers on "Protein maturation published in 2021"

Soluble Programmed Death Ligand-1 (sPD-L1): A Pool of Circulating Proteins Implicated in Health and Diseases.

Abstract: Upon T-cell receptor stimulation, the Programmed cell Death-1 receptor (PD-1) expressed on T-cells can interact with its ligand PD-L1 expressed at the surface of cancer cells or antigen-presenting cells. Monoclonal antibodies targeting PD-1 or PD-L1 are routinely used for the treatment of cancers, but their clinical efficacy varies largely across the variety of tumor types. A part of the variability is linked to the existence of several forms of PD-L1, either expressed on the plasma membrane (mPD-L1), at the surface of secreted cellular exosomes (exoPD-L1), in cell nuclei (nPD-L1), or as a circulating, soluble protein (sPD-L1). Here, we have reviewed the different origins and roles of sPD-L1 in humans to highlight the biochemical and functional heterogeneity of the soluble protein. sPD-L1 isoforms can be generated essentially by two non-exclusive processes: (i) proteolysis of m/exoPD-L1 by metalloproteases, such as metalloproteinases (MMP) and A disintegrin and metalloproteases (ADAM), which are capable of shedding membrane PD-L1 to release an active soluble form, and (ii) the alternative splicing of PD-L1 pre-mRNA, leading in some cases to the release of sPD-L1 protein isoforms lacking the transmembrane domain. The expression and secretion of sPD-L1 have been observed in a large variety of pathologies, well beyond cancer, notably in different pulmonary diseases, chronic inflammatory and autoimmune disorders, and viral diseases. The expression and role of sPD-L1 during pregnancy are also evoked. The structural heterogeneity of sPD-L1 proteins, and associated functional/cellular plurality, should be kept in mind when considering sPD-L1 as a biomarker or as a drug target. The membrane, exosomal and soluble forms of PD-L1 are all integral parts of the highly dynamic PD-1/PD-L1 signaling pathway, essential for immune-tolerance or immune-escape.

Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.

Abstract: Heat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s) As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37 Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways Human sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation Human sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38 Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation Taken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways

Mechanisms of Cotranslational Protein Maturation in Bacteria.

Abstract: Growing cells invest a significant part of their biosynthetic capacity into the production of proteins. To become functional, newly-synthesized proteins must be N-terminally processed, folded and often translocated to other cellular compartments. A general strategy is to integrate these protein maturation processes with translation, by cotranslationally engaging processing enzymes, chaperones and targeting factors with the nascent polypeptide. Precise coordination of all factors involved is critical for the efficiency and accuracy of protein synthesis and cellular homeostasis. This review provides an overview of the current knowledge on cotranslational protein maturation, with a focus on the production of cytosolic proteins in bacteria. We describe the role of the ribosome and the chaperone network in protein folding and how the dynamic interplay of all cotranslationally acting factors guides the sequence of cotranslational events. Finally, we discuss recent data demonstrating the coupling of protein synthesis with the assembly of protein complexes and end with a brief discussion of outstanding questions and emerging concepts in the field of cotranslational protein maturation.

Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene.

Abstract: Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

Abstract: Murine xenografts of pediatric leukemia accurately recapitulate genomic aberrations. How this translates to the functional capacity of cells remains unclear. Here, we studied global protein abundance, phosphorylation, and protein maturation by proteolytic processing in 11 pediatric B- and T- cell ALL patients and 19 corresponding xenografts. Xenograft models were generated for each pediatric patient leukemia. Mass spectrometry-based methods were used to investigate global protein abundance, protein phosphorylation, and limited proteolysis in paired patient and xenografted pediatric acute B- and T- cell lymphocytic leukemia, as well as in pediatric leukemia cell lines. Targeted next-generation sequencing was utilized to examine genetic abnormalities in patients and in corresponding xenografts. Bioinformatic and statistical analysis were performed to identify functional mechanisms associated with proteins and protein post-translational modifications. Overall, we found xenograft proteomes to be most equivalent with their patient of origin. Protein level differences that stratified disease subtypes at diagnostic and relapse stages were largely recapitulated in xenografts. As expected, PDXs lacked multiple human leukocyte antigens and complement proteins. We found increased expression of cell cycle proteins indicating a high proliferative capacity of xenografted cells. Structural genomic changes and mutations were reflected at the protein level in patients. In contrast, the post-translational modification landscape was shaped by leukemia type and host and only to a limited degree by the patient of origin. Of 201 known pediatric oncogenic drivers and drug-targetable proteins, the KMT2 protein family showed consistently high variability between patient and corresponding xenografts. Comprehensive N terminomics revealed deregulated proteolytic processing in leukemic cells, in particular from caspase-driven cleavages found in patient cells. Genomic and host factors shape protein and post-translational modification landscapes differently. This study highlights select areas of diverging biology while confirming murine patient-derived xenografts as a generally accurate model system.

Biallelic mutations in L-dopachrome tautomerase (DCT) cause infantile nystagmus and oculocutaneous albinism.

Abstract: Infantile nystagmus syndrome (INS) denominates early-onset, involuntary oscillatory eye movements with different etiologies. Nystagmus is also one of the symptoms in oculocutaneus albinism (OCA), a heterogeneous disease mainly caused by defects in melanin synthesis or melanosome biogenesis. Dopachrome tautomerase (DCT, also called TYRP2) together with tyrosinase (TYR) and tyrosin-related protein 1 (TYRP1) is one of the key enzymes in melanin synthesis. Although DCT´s role in pigmentation has been proven in different species, until now only mutations in TYR and TYRP1 have been found in patients with OCA. Detailed ophthalmological and orthoptic investigations identified a consanguineous family with two individuals with isolated infantile nystagmus and one family member with subtle signs of albinism. By whole-exome sequencing and segregation analysis, we identified the missense mutation c.176G > T (p.Gly59Val) in DCT in a homozygous state in all three affected family members. We show that this mutation results in incomplete protein maturation and targeting in vitro compatible with a partial or total loss of function. Subsequent screening of a cohort of patients with OCA (n = 85) and INS (n = 25) revealed two heterozygous truncating mutations, namely c.876C > A (p.Tyr292*) and c.1407G > A (p.Trp469*), in an independent patient with OCA. Taken together, our data suggest that mutations in DCT can cause a phenotypic spectrum ranging from isolated infantile nystagmus to oculocutaneous albinism.

Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

Abstract: The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions.

RNA sequencing-based analysis of the magnum tissues revealed the novel genes and biological pathways involved in the egg-white formation in the laying hen.

Abstract: The mechanism of egg formation in the oviduct of laying hens is tightly controlled; each segment of the oviduct contributes a unique component of the egg. Several genes/proteins are involved in the synthesis of a completely healthy egg. This implies a time- and tissue-specific expression of genes and proteins in the different oviductal segments. We used hens at different physiological stages and time points to understand the transcriptional regulation of egg-white (albumen) synthesis and secretion onto the eggs in the magnum of laying hens. This study used Next-Generation Sequencing and quantitative real-time PCR (qPCR) to detect the novel genes and the cognate biological pathways that regulate the major events during the albumen formation. Magnum tissues collected from laying (n = 5 each at 3 h post-ovulation, p.o. and 15–20 h p.o.), non-laying (n = 4), and molting (n = 5) hens were used for differential gene expression analyses. A total of 540 genes (152 upregulated and 388 down-regulated) were differentially expressed at 3 h p.o. in the magnum of laying hens. Kyoto Encyclopedia of Genes and Genomes pathways analysis of the 152 upregulated genes revealed that glycine, serine, and threonine metabolism was the most-enriched biological pathway. Furthermore, the top two most enriched keywords for the upregulated genes were amino-acid biosynthesis and proteases. Nine candidate genes associated with albumen formation were validated with qPCR to have differential expression in laying, non-laying, and molting hens. Proteases such as TMPRSS9, CAPN2, MMP1, and MMP9 (protein maturation, ECM degradation, and angiogenesis); enzymes such as PSPH, PHGDH, and PSAT1 (amino-acid biosynthesis); RLN3, ACE, and REN (albumen synthesis, secretion and egg transport); and AVD, AvBD11, and GPX3 (antimicrobial and antioxidants) were recognized as essential molecules linked to albumen deposition in the magnum. This study revealed some novel genes that participate in the signaling pathways for egg-white synthesis and secretion along with some well-known functional genes. These findings help to understand the mechanisms involved in albumen biosynthesis.

How Is the Fidelity of Proteins Ensured in Terms of Both Quality and Quantity at the Endoplasmic Reticulum? Mechanistic Insights into E3 Ubiquitin Ligases.

Abstract: The endoplasmic reticulum (ER) is an interconnected organelle that plays fundamental roles in the biosynthesis, folding, stabilization, maturation, and trafficking of secretory and transmembrane proteins. It is the largest organelle and critically modulates nearly all aspects of life. Therefore, in the endoplasmic reticulum, an enormous investment of resources, including chaperones and protein folding facilitators, is dedicated to adequate protein maturation and delivery to final destinations. Unfortunately, the folding and assembly of proteins can be quite error-prone, which leads to the generation of misfolded proteins. Notably, protein homeostasis, referred to as proteostasis, is constantly exposed to danger by flows of misfolded proteins and subsequent protein aggregates. To maintain proteostasis, the ER triages and eliminates terminally misfolded proteins by delivering substrates to the ubiquitin-proteasome system (UPS) or to the lysosome, which is termed ER-associated degradation (ERAD) or ER-phagy, respectively. ERAD not only eliminates misfolded or unassembled proteins via protein quality control but also fine-tunes correctly folded proteins via protein quantity control. Intriguingly, the diversity and distinctive nature of E3 ubiquitin ligases determine efficiency, complexity, and specificity of ubiquitination during ERAD. ER-phagy utilizes the core autophagy machinery and eliminates ERAD-resistant misfolded proteins. Here, we conceptually outline not only ubiquitination machinery but also catalytic mechanisms of E3 ubiquitin ligases. Further, we discuss the mechanistic insights into E3 ubiquitin ligases involved in the two guardian pathways in the ER, ERAD and ER-phagy. Finally, we provide the molecular mechanisms by which ERAD and ER-phagy conduct not only protein quality control but also protein quantity control to ensure proteostasis and subsequent organismal homeostasis.

New insights into the role of endoplasmic reticulum-associated degradation in Bartter Syndrome Type 1.

Abstract: Mutations in Na-K-2Cl co-transporter, NKCC2, lead to type I Bartter syndrome (BS1), a life-threatening kidney disease. Yet, our knowledge of the molecular regulation of NKCC2 mutants remains poor. Here, we aimed to identify the molecular pathogenic mechanisms of one novel and three previously reported missense NKCC2 mutations. Co-immunolocalization studies revealed that all NKCC2 variants are not functional because they are not expressed at the cell surface due to retention in the endoplasmic reticulum. Cycloheximide chase assays together with treatment by protein degradation and mannose trimming inhibitors demonstrated that the defect in NKCC2 maturation arise from ER retention and associated degradation (ERAD). siRNA knock-down experiments revealed that the ER lectin OS9 is involved in the ERAD of NKCC2 mutants. 4-PBA treatment mimicked OS9 knock-down effect on NKCC2 mutants by stabilizing their immature forms. Importantly, out of the four studied mutants, only one showed an increased protein maturation upon treatment with glycerol. In summary, our study reveals that BS1 is among diseases linked to the ERAD pathway. Moreover, our data open the possibility that maturation of some ER retained NKCC2 variants is correctable by chemical chaperones offering therefore promising avenues in elucidating the molecular pathways governing the ERAD of NKCC2 folding mutants. This article is protected by copyright. All rights reserved.

QM/MM Approaches Shed Light on GFP Puzzles

Abstract: The green fluorescent protein (GFP) is one of the most important light-responsive biological systems. We describe applications of a particular QM/MM version based on the flexible effective fragment potential theory to characterize the properties of this protein including transformations between different forms of GFP. We also show how the chemical reactions of chromophore maturation in GFP and the photochemical reactions leading to GFP malfunction are characterized in QM/MM simulations.

PPARγ activation in late gestation does not promote surfactant maturation in the fetal sheep lung

Abstract: Respiratory distress syndrome results from inadequate functional pulmonary surfactant and is a significant cause of mortality in preterm infants. Surfactant is essential for regulating alveolar interfacial surface tension, and its synthesis by Type II alveolar epithelial cells is stimulated by leptin produced by pulmonary lipofibroblasts upon activation by peroxisome proliferator-activated receptor γ (PPARγ). As it is unknown whether PPARγ stimulation or direct leptin administration can stimulate surfactant synthesis before birth, we examined the effect of continuous fetal administration of either the PPARγ agonist, rosiglitazone (RGZ; Study 1) or leptin (Study 2) on surfactant protein maturation in the late gestation fetal sheep lung. We measured mRNA expression of genes involved in surfactant maturation and showed that RGZ treatment reduced mRNA expression of LPCAT1 (surfactant phospholipid synthesis) and LAMP3 (marker for lamellar bodies), but did not alter mRNA expression of PPARγ, surfactant proteins (SFTP-A, -B, -C, and -D), PCYT1A (surfactant phospholipid synthesis), ABCA3 (phospholipid transportation), or the PPARγ target genes SPHK-1 and PAI-1. Leptin infusion significantly increased the expression of PPARγ and IGF2 and decreased the expression of SFTP-B. However, mRNA expression of the majority of genes involved in surfactant synthesis was not affected. These results suggest a potential decreased capacity for surfactant phospholipid and protein production in the fetal lung after RGZ and leptin administration, respectively. Therefore, targeting PPARγ may not be a feasible mechanistic approach to promote lung maturation.

Genetic Approaches to Uncover Gene Products Involved in Iron-Sulfur Protein Maturation: High-Throughput Genomic Screening Using Transposon Sequencing.

Abstract: Iron-sulfur (Fe-S) clusters are one of the most ubiquitous and versatile prosthetic groups exploited by nature. Fe-S clusters aid in conducting redox reactions, carbon activation, and environmental sensing. This chapter presents an overview of the genetic approaches that have been useful for identifying and characterizing bacterial factors involved in Fe-S protein assembly. Traditional genetic screens that assess viability or conditional auxotrophies and bioinformatic approaches have identified the majority of the described genes utilized for Fe-S protein assembly. Herein, we expand upon this list of genetic methods by detailing the use of transposon sequencing (TnSeq) to identify gene products that are necessary for the proper function of metabolic pathways that require Fe-S enzymes. TnSeq utilizes the power of genomics and massively parallel DNA sequencing to allow researchers to quantify the necessity of individual gene products for a specific growth condition. This allows for the identification of gene products or gene networks that have a role in a given metabolic process but are not essential for the process. An advantage of this approach is that it allows researchers to identify mutants that have partial phenotypes that are often missed using traditional plate-based selections. Applying TnSeq to address questions of Fe-S protein maturation will result in a more comprehensive understanding of genetic interactions and factors utilized in Fe-S biogenesis and Fe-S protein assembly.

Computational Modeling of C-Terminal Tails to Predict the Calcium-Dependent Secretion of Endoplasmic Reticulum Resident Proteins

Abstract: The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention/retrieval sequence (ERS). The canonical ERS that promotes ER retrieval is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical “KDEL” ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interacts with the KDELRs. The interaction between the ERS and the KDELR extends beyond the final four carboxy terminal residues of the ERS. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

CFTR corrector efficacy is associated with occupancy of distinct binding sites

Abstract: CFTR misfolding due to cystic fibrosis causing mutations can be corrected with small molecules designated as correctors. VX-809, an investigational corrector compound, is believed to bind CFTR directly to either the first membrane-spanning domain (MSD1) and/or the first nucleotide-binding domain (NBD1). Blind docking onto the 3D structures of these domains, followed by molecular dynamics (MD) simulations, revealed the presence of two potential VX-809 binding sites which, when mutated, abrogated corrector rescue. Mutations altering protein maturation are also shown to be not equally sensitive to the occupancy of the two sites by VX-809, with the most frequent mutation F508del requiring integrity of both sites and allosteric coupling with the F508del region while L206W only requires the integrity of the MSD1 site. A network of charged amino acids in the lasso Lh2 helix and the intracellular loops ICL1 and ICL4 is involved in the allostery between MSD1 and NBD1. Corrector VX-445, which is used in combination in clinics with VX-661, a structurally close analog of VX-809, to fully correct F508del, is also shown to occupy two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. In conclusion, VX-809 and VX-445 appear to bind different CFTR domains to alleviate specific folding defects. These results provide new insights into therapeutics understanding and may help the development of efficient corrector combinations.

Von Willebrand Factor Facilitates Intravascular Dissemination of Microsporidia Encephalitozoon hellem.

Abstract: Microsporidia are a group of spore-forming, fungus-related pathogens that can infect both invertebrates and vertebrates including humans. The primary infection site is usually digestive tract, but systemic infections occur as well and cause damages to organs such as lung, brain, and liver. The systemic spread of microsporidia may be intravascular, requiring attachment and colonization in the presence of shear stress. Von Willebrand Factor (VWF) is a large multimeric intravascular protein and the key attachment sites for platelets and coagulation factors. Here in this study, we investigated the interactions between VWF and microsporidia Encephalitozoon hellem (E. hellem), and the modulating effects on E. hellem after VWF binding. Microfluidic assays showed that E. hellem binds to ultra-large VWF strings under shear stress. In vitro germination assay and infection assay proved that E. hellem significantly increased the rates of germination and infection, and these effects would be reversed by VWF blocking antibody. Mass spectrometry analysis further revealed that VWF-incubation altered various aspects of E. hellem including metabolic activity, levels of structural molecules, and protein maturation. Our findings demonstrated that VWF can bind microsporidia in circulation, and modulate its pathogenicity, including promoting germination and infection rate. VWF facilitates microsporidia intravascular spreading and systemic infection.

Computational modeling of C-terminal tails to predict the calcium-dependent secretion of ER resident proteins

Abstract: The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention sequence (ERS). The canonical ERS is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is also regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical “KDEL” ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interact with the KDELRs. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

SARS-CoV-2 Delta Spike Protein Enhances the Viral Fusogenicity and Inflammatory Cytokine Production (preprint)

Abstract: The Delta variant is now the most dominant and virulent SARS-CoV-2 variant of concern (VOC). In this study, we investigated several virological features of Delta spike protein (SPDelta), including protein maturation and its impact on viral entry of cell-free pseudotyped virus, cell-cell fusion ability and its induction of inflammatory cytokine production in human macrophages and dendritic cells. The results showed that SPΔCDelta exhibited enhanced S1/S2 cleavage in cells and pseudotyped virus-like particles (PVLPs). We further showed that SPΔCDelta elevated pseudovirus infection in human lung cell lines and mediated significantly enhanced syncytia formation. Furthermore, we revealed that SPΔCDelta-PVLPs had stronger effects on stimulating NF-κB and AP-1 signaling in human monocytic THP1 cells and induced significantly higher levels of pro-inflammatory cytokine, such as TNF-α, IL-1β and IL-6, released from human macrophages and dendritic cells. Overall, these studies provide evidence to support the important role of SPΔCDelta during virus infection, transmission and pathogenesis.

Computational modeling of C-terminal tails to predict the calcium-dependent secretion of ER resident proteins

Abstract: The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention sequence (ERS). The canonical ERS is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is also regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical “KDEL” ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interact with the KDELRs. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

Molecular Characterization and Expression of the LAP3 Gene and Its Association with Growth Traits in the Blood Clam Tegillarca granosa

Abstract: Leucine aminopeptidase 3 (LAP3) is a metallopeptidase that cleaves N-terminal residues and is involved in protein maturation and degradation. In this study, we characterized the leucine aminopeptidase 3 (LAP3) gene from Tegillarca granosa (Tg-LAP3 for short), which appeared to consist of 15,731 nucleotides encoding 530 amino acids. We identified 12 introns and 13 exons in the Tg-LAP3 gene, suggesting a highly conserved genomic structure. The proximal promoter sequence consists of 1922 bps with a typical TATA box structure, which is the general structural characteristic of core promoters in eukaryotes. We found two functional domains in the Tg-LAP3 protein, including an N-terminal domain (41–174aa) and a peptidase_M17 catalytic domain (209–522aa). Multiple alignment showed that Tg-LAP3 shares 73.4% identity with LAP3 of Mizuhopecten yessoensis and 55.2–70.7% identity with LAP3 of other species. Quantitative analysis of Tg-LAP3 in embryos/larvae and adult tissues indicated that the highest expression occurred in eyebot larva, with limited expression in other stages; among tissues, the highest expression was found in the liver (p G, g.-1123C > T, and g.-1304C > A) in the proximal promoter were successfully typed, but there was no significant difference in growth traits (body weight, shell length, shell width, and shell height) among these genotypes. The results of our study demonstrate the functional roles of the Tg-LAP3 gene and provide valuable information for molecular marker-assisted selection (MAS) of the blood clam.

Bacterial Approaches for Assembling Iron-Sulfur Proteins.

Abstract: Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target.