Regulation Of Protein Function Research Articles

A Layered Genetic Design Enables the Yeast Galactose Regulon to Respond to Cyanamide.

The commonly used expression systems in Saccharomyces cerevisiae typically rely on either constitutive or galactose-regulated promoters. The lack of inducible systems in S.cerevisiae limits the precise temporal regulation of protein function and yeast metabolism. We herein repurposed the galactose-regulated system to make it respond to cyanamide. By using a cyanamide-inducible DDI2 promoter to control Gal4 expression in CEN.PK2-1C with Δgal80, a tight and graded cyanamide-inducible GAL system with an enhanced signal output was constructed. Subsequently, we demonstrated that the cyanamide-inducible GAL system was capable of tightly regulating the pentafunctional Aro1 protein to achieve conditional shikimate pathway activity. Taken together, the cyanamide-inducible GAL system could be implemented for both fundamental research and applied biotechnology.

Functional Diversity of SIRT7 Across Cellular Compartments: Insights and Perspectives.

Posttranslational modifications (PTMs) play important roles in the regulation of protein function. Acetylation and deacetylation are among the most important PTMs. SIRT7 is a relatively understudied member of the sirtuin family, but recent studies have revealed that it plays a regulatory role in a variety of cellular activities, such as genome stabilization and repair, gene translation, ribosome production and other important processes. Here, we provide a list of the functions and mechanisms of SIRT7 in various organelles and show the important role of SIRT7 in maintaining normal cell function.

Editorial: Post-transcriptional regulation of viral protein expression and function

Editorial: Post-transcriptional regulation of viral protein expression and function

Abstract P2008.1: Association Of Myocardial Stiffening With Impaired Relaxation In A Diabetic Murine Model Of Left Ventricular Diastolic Dysfunction

Introduction: Left ventricular diastolic dysfunction (LVDD) is often manifested by impaired relaxation and myocardial stiffening in the left ventricle (LV). While impairments in the function of myofilament regulatory proteins are known to deteriorate LV relaxation, the effects of passive myocardial stiffening on the relaxation of active forces remain unknown. Methods: A diabetic murine model of LVDD (db/db, n=5) and wild-type mice (WT, n=5) were used to study the association between tissue-level passive stiffness and organ-level relaxation. Early transmitral flow velocity to mitral annular tissue velocity ratio (E/e’), measured by Doppler imaging, was used to assess LV relaxation. Cardiac magnetic resonance imaging was performed using a 7T Bruker scanner to estimate diastolic strains via image registration. Mechanical testing was performed on harvested LV free wall (LVFW) specimens to measure passive stiffness. Results: LVDD was confirmed by a high septal E/e’ ratio (Fig. 1A). The db/db mice presented an increase in passive LVFW stiffness measured as the tangent to the circumferential stress-strain curve at 30% strain (Fig. 1B). A positive correlation was observed between passive stiffness and E/e’ (Fig. 1C). End-diastolic strain calculations (Fig. 1D) corroborated LV filling impairment indicating significant reductions in circumferential LV strains (Fig. 1E). Conclusion: Passive stiffening may impair LV filling by not only restraining the passive stretch of the myocardium at diastole but also by decelerating the active relaxation. Further integrated in-vivo and ex-vivo studies of LVDD can deconvolute the “active” and “passive” causes of impaired relaxation.

Impact of Protein Nα-Modifications on Cellular Functions and Human Health.

Most human proteins are modified by enzymes that act on the α-amino group of a newly synthesized polypeptide. Methionine aminopeptidases can remove the initiator methionine and expose the second amino acid for further modification by enzymes responsible for myristoylation, acetylation, methylation, or other chemical reactions. Specific acetyltransferases can also modify the initiator methionine and sometimes the acetylated methionine can be removed, followed by further modifications. These modifications at the protein N-termini play critical roles in cellular protein localization, protein-protein interaction, protein-DNA interaction, and protein stability. Consequently, the dysregulation of these modifications could significantly change the development and progression status of certain human diseases. The focus of this review is to highlight recent progress in our understanding of the roles of these modifications in regulating protein functions and how these enzymes have been used as potential novel therapeutic targets for various human diseases.

Cooperativity in regulation of membrane protein function: phenomenological analysis of the effects of pH and phospholipids.

Interaction between membrane proteins and ligands plays a key role in governing a wide spectrum of cellular processes. These interactions can provide a cooperative-type regulation of protein function. A wide variety of proteins, including enzymes, channels, transporters, and receptors, displays cooperative behavior in their interactions with ligands. Moreover, the ligands involved encompass a vast diversity and include specific molecules or ions that bind to specific binding sites. In this review, our particular focus is on the interaction between integral membrane proteins and ligands that can present multiple"binding sites", such as protons or membrane phospholipids. The study of the interaction that protons or lipids have with membrane proteins often presents challenges for classical mechanistic modeling approaches. In this regard, we show that, like Hill's pioneering work on hemoglobin regulation, phenomenological modeling constitutes a powerful tool for capturing essential features of these systems.

Core fucosylation and its roles in gastrointestinal glycoimmunology.

Glycosylation is a common post-translational modification in eukaryotic cells. It is involved in the production of many biologically active glycoproteins and the regulation of protein structure and function. Core fucosylation plays a vital role in the immune response. Most immune system molecules are core fucosylated glycoproteins such as complements, cluster differentiation antigens, immunoglobulins, cytokines, major histocompatibility complex molecules, adhesion molecules, and immune molecule synthesis-related transcription factors. These core fucosylated glycoproteins play important roles in antigen recognition and clearance, cell adhesion, lymphocyte activation, apoptosis, signal transduction, and endocytosis. Core fucosylation is dominated by fucosyltransferase 8 (Fut8), which catalyzes the addition of α-1,6-fucose to the innermost GlcNAc residue of N-glycans. Fut8 is involved in humoral, cellular, and mucosal immunity. Tumor immunology is associated with aberrant core fucosylation. Here, we summarize the roles and potential modulatory mechanisms of Fut8 in various immune processes of the gastrointestinal system.

Proline Isomerization: From the Chemistry and Biology to Therapeutic Opportunities.

Proline isomerization, the process of interconversion between the cis- and trans-forms of proline, is an important and unique post-translational modification that can affect protein folding and conformations, and ultimately regulate protein functions and biological pathways. Although impactful, the importance and prevalence of proline isomerization as a regulation mechanism in biological systems have not been fully understood or recognized. Aiming to fill gaps and bring new awareness, we attempt to provide a wholistic review on proline isomerization that firstly covers what proline isomerization is and the basic chemistry behind it. In this section, we vividly show that the cause of the unique ability of proline to adopt both cis- and trans-conformations in significant abundance is rooted from the steric hindrance of these two forms being similar, which is different from that in linear residues. We then discuss how proline isomerization was discovered historically followed by an introduction to all three types of proline isomerases and how proline isomerization plays a role in various cellular responses, such as cell cycle regulation, DNA damage repair, T-cell activation, and ion channel gating. We then explore various human diseases that have been linked to the dysregulation of proline isomerization. Finally, we wrap up with the current stage of various inhibitors developed to target proline isomerases as a strategy for therapeutic development.

Targeting transcriptional kinase of CDK7 halts proliferation of multiple myeloma cells by modulating the function of canonical NF-kB pathway and cell cycle regulatory proteins

Multiple myeloma (MM) is an incurable plasma cell neoplasm. Despite several effective frontline therapeutic regimens, including Bortezomib (BTZ), relapse is almost inevitable; therefore, better therapeutic modalities to improve the outcomes are needed. Cyclin-dependent kinases (CDKs) are an essential constituent of the cellular transcriptional machinery and tumors including MM are critically dependent on transcription to maintain their oncogenic state. In the present study, we explored the efficacy of THZ1, a covalent CDK7 inhibitor in MM treatment using Bortezomib resistant (H929BTZR) cells and zebrafish xenografts. THZ1 showed anti-myeloma activity in the models of MM but had no effect on healthy CD34+ cells. THZ1 suppresses phosphorylation of carboxy-terminal domain of RNA polymerase II and downregulates the transcription of BCL2 family of proteins both in H929BTZS and H929BTZR cells leading to G1/S arrest and apoptosis. THZ1 mediates inhibition of bone marrow stromal cells-induced proliferation and activation of NF-kB signaling. The data derived from zebrafish xenografts of MM demonstrate that THZ1 combined with BTZ synergistically reduces tumor growth in zebrafish embryos. Collectively, our results reveal that THZ1 alone as well as in combination with BTZ has effective anti-myeloma activity.

O-Glycopeptide Truncation Strategy for Heterogeneous O-GalNAc Glycoproteomics Characterization.

Mucin-type O-glycosylation (or O-GalNAcylation) takes place on most membrane and secretory proteins and is vital in regulating protein functions and many biological processes. O-GalNAcylation generally exhibits highly diverse and dense O-glycans linked to carrier proteins, which challenges the analysis of O-GalNAc glycoproteome using conventional methodologies. Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes proteases or O-glycopeptidases for targeted cleavage of the enriched tryptic O-glycopeptides. It simplifies the O-glycopeptide backbones, O-glycans, or both, and has been shown to aid the improvement of the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. The enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for mass spectrometry detection and database search in general bottom-up glycoproteomics. We also investigate different proteolysis which could be well integrated into the O-glycopeptide truncation strategy. For large-scale analysis, we exploit different truncation schemes and identify nearly 2000 O-glycopeptides corresponding to 391 glycoproteins from 75 μL human serum, achieving the deepest-scale coverage of O-glycoproteins compared to other plasma/serum O-glycoproteomic studies. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.

The discovery, function, and regulation of epithelial splicing regulatory proteins (ESRP) 1 and 2.

Alternative splicing is a broad and evolutionarily conserved mechanism to diversify gene expression and functionality. The process relies on RNA binding proteins (RBPs) to recognize and bind target sequences in pre-mRNAs, which allows for the inclusion or skipping of various alternative exons. One recently discovered family of RBPs is the epithelial splicing regulatory proteins (ESRP) 1 and 2. Here, we discuss the structure and physiological function of the ESRPs in a variety of contexts. We emphasize the current understanding of their splicing activities, using the classic example of fibroblast growth factor receptor 2 mutually exclusive splicing. We also describe the mechanistic roles of ESRPs in coordinating the splicing and functional output of key signaling pathways that support the maintenance of, or shift between, epithelial and mesenchymal cell states. In particular, we highlight their functions in the development of mammalian limbs, the inner ear, and craniofacial structure while discussing the genetic and biochemical evidence that showcases their conserved roles in tissue regeneration, disease, and cancer pathogenesis.

BiGRUD-SA: Protein S-sulfenylation sites prediction based on BiGRU and self-attention

S-sulfenylation is a vital post-translational modification (PTM) of proteins, which is an intermediate in other redox reactions and has implications for signal transduction and protein function regulation. However, there are many restrictions on the experimental identification of S-sulfenylation sites. Therefore, predicting S-sulfoylation sites by computational methods is fundamental to studying protein function and related biological mechanisms. In this paper, we propose a method named BiGRUD-SA based on bi-directional gated recurrent unit (BiGRU) and self-attention mechanism to predict protein S-sulfenylation sites. We first use AAC, BLOSUM62, AAindex, EAAC and GAAC to extract features, and do feature fusion to obtain original feature space. Next, we use SMOTE-Tomek method to handle data imbalance. Then, we input the processed data to the BiGRU and use self-attention mechanism to do further feature extraction. Finally, we input the data obtained to the deep neural networks (DNN) to identify S-sulfenylation sites. The accuracies of training set and independent test set are 96.66% and 95.91% respectively, which indicates that our method is conducive to identifying S-sulfenylation sites. Furthermore, we use a data set of S-sulfenylation sites in Arabidopsis thaliana to effectively verify the generalization ability of BiGRUD-SA method, and obtain better prediction results.

Adenosine A2A Receptor Activation Regulates Niemann-Pick C1 Expression and Localization in Macrophages.

Adenosine plays an important role in modulating immune cell function, particularly T cells and myeloid cells, such as macrophages and dendritic cells. Cell surface adenosine A2A receptors (A2AR) regulate the production of pro-inflammatory cytokines and chemokines, as well as the proliferation, differentiation, and migration of immune cells. In the present study, we expanded the A2AR interactome and provided evidence for the interaction between the receptor and the Niemann-Pick type C intracellular cholesterol transporter 1 (NPC1) protein. The NPC1 protein was identified to interact with the C-terminal tail of A2AR in RAW 264.7 and IPMФ cells by two independent and parallel proteomic approaches. The interaction between the NPC1 protein and the full-length A2AR was further validated in HEK-293 cells that permanently express the receptor and RAW264.7 cells that endogenously express A2AR. A2AR activation reduces the expression of NPC1 mRNA and protein density in LPS-activated mouse IPMФ cells. Additionally, stimulation of A2AR negatively regulates the cell surface expression of NPC1 in LPS-stimulated macrophages. Furthermore, stimulation of A2AR also altered the density of lysosome-associated membrane protein 2 (LAMP2) and early endosome antigen 1 (EEA1), two endosomal markers associated with the NPC1 protein. Collectively, these results suggested a putative A2AR-mediated regulation of NPC1 protein function in macrophages, potentially relevant for the Niemann-Pick type C disease when mutations in NPC1 protein result in the accumulation of cholesterol and other lipids in lysosomes.

Impact of transmembrane peptides on individual lipid motions and collective dynamics of lipid bilayers

The fluid nature of lipid bilayers is indispensable for the dynamic regulation of protein function and membrane morphology in biological membranes. Membrane-spanning domains of proteins interact with surrounding lipids and alter the physical properties of lipid bilayers. However, there is no comprehensive view of the effects of transmembrane proteins on the membrane's physical properties. Here, we investigated the effects of transmembrane peptides with different flip-flop-promoting abilities on the dynamics of a lipid bilayer employing complemental fluorescence and neutron scattering techniques. The quasi-elastic neutron scattering and fluorescence experiments revealed that lateral diffusion of the lipid molecules and the acyl chain motions were inhibited by the inclusion of transmembrane peptides. The neutron spin-echo spectroscopy measurements indicated that the lipid bilayer became more rigid but more compressible and the membrane viscosity increased when the transmembrane peptides were incorporated into the membrane. These results suggest that the inclusion of rigid transmembrane structures hinders individual and collective lipid motions by slowing down lipid diffusion and increasing interleaflet coupling. The present study provides a clue for understanding how the local interactions between lipids and proteins change the collective dynamics of the lipid bilayers, and therefore, the function of biological membranes.

Large‐scale phosphomimetic screening identifies phospho‐modulated motif‐based protein interactions

Phosphorylation is a ubiquitous post‐translation modification that regulates protein function by promoting, inhibiting or modulating protein–protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide‐phage display library to screen for phosphosites that modulate short linear motif‐based interactions. The peptidome covers ~13,500 phospho‐serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild‐type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif‐mediated interactions. Affinity measurements confirmed the phospho‐modulation of 14 out of 18 tested interactions. We performed a detailed follow‐up on a phospho‐dependent interaction between clathrin and the mitotic spindle protein hepatoma‐upregulated protein (HURP), demonstrating the essentiality of the phospho‐dependency to the mitotic function of HURP. Structural characterisation of the clathrin‐HURP complex elucidated the molecular basis for the phospho‐dependency. Our work showcases the power of phosphomimetic ProP‐PD to discover novel phospho‐modulated interactions required for cellular function.

Deletion of Brucella transcriptional regulator GntR10 regulated the expression of quorum sensing system and type IV secretion system effectors, which affected the activation of NF-κB

GntR10 is a transcriptional regulator in Brucella. Nuclear factor-kappa B (NF-κB) is involved in many cellular activities, playing major roles in orchestrating the expression of inflammatory genes and regulating protein function that is essential for pathogenic bacteria during infection. GntR10 deletion was previously found to affect the growth and the virulence of Brucella and expression levels of target genes of GntR10 in mice. However, the mechanisms of affection of NF-κB regulated by Brucella GntR10 are still unclear. Here, GntR10 deletion could regulate the expression of LuxR-type transcriptional activators (VjbR and BlxR) of the quorum sensing system (QSS) and type IV secretion system (T4SS) effectors (BspE and BspF) of Brucella. It could further inhibit the activation of the regulator NF-κB and affect the virulence of Brucella. This research provides new insights into the designing of Brucella vaccines and the screening of drug targets. SignificanceTranscriptional regulators are predominant bacterial signal transduction factors. The pathogenicity of Brucella is due to its ability to regulate the expression of virulence related genes including quorum sensing system (QSS) and type IV secretion system (T4SS). Transcriptional regulators are designed to regulate gene expression and enact an appropriate adaptive physiological response. Here, we show that Brucella transcriptional regulator GntR10 regulated the expression of QSS and T4SS effectors, which affected the activation of NF-κB.

Photocaged activity-based probes for improved monitoring of protein S-sulfenylation in living cells

Protein S-sulfenylation (protein sulfenic acid), as one of the most significant oxidative post-translational modifications (OxiPTMs), plays a vital role in regulating protein function. A variety of activity-based probes have been developed to profile sulfenic acid in living cells. However, due to the transient presence and low content of sulfenic acid in living cell, high doses of probes are needed to achieve efficient labeling. More importantly, current probes have no temporal control over sulfenic acid labeling. To overcome these limitations, two caged cysteine sulfenic acid probes DYn-2-ONB and DYn-2-Cou with either an o-nitrobenzyl or coumarin protecting group were developed in this study. Both probes can be efficiently uncaged via irradiation to produce the active C-nucleophile probe DYn-2. Labeling assay in living cells demonstrated DYn-2-ONB exhibited better labeling capacity compared with DYn-2, providing it as a powerful tool for improved monitoring of protein S-sulfenylation in living cells.

Protein Persulfidation: Recent Progress and Future Directions.

Significance: Hydrogen sulfide (H2S) is considered to be a gasotransmitter along with carbon monoxide (CO) and nitric oxide (NO), and is known as a key regulator of physiological and pathological activities. S-sulfhydration (also known as persulfidation), a mechanism involving the formation of protein persulfides by modification of cysteine residues, is proposed here to explain the multiple biological functions of H2S. Investigating the properties of protein persulfides can provide a foundation for further understanding of the potential functions of H2S. Recent Advances: Multiple methods have been developed to determine the level of protein persulfides. It has been demonstrated that protein persulfidation is involved in many biological processes through various mechanisms including the regulation of ion channels, enzymes, and transcription factors, as well as influencing protein-protein interactions. Critical Issues: Some technical and theoretical questions remain to be solved. These include how to improve the specificity of the detection methods for protein persulfidation, why persulfidation typically occurs on one or a few thiols within a protein, how this modification alters protein functions, and whether protein persulfidation has organ-specific patterns. Future Directions: Optimizing the detection methods and elucidating the properties and molecular functions of protein persulfidation would be beneficial for current therapeutics. In this review, we introduce the detailed mechanism of the persulfidation process and discuss persulfidation detection methods. In addition, this review summarizes recent discoveries of the selectivity of protein persulfidation and the regulation of protein functions and cell signaling pathways by persulfidation. Antioxid. Redox Signal. 39, 829-852.

Abstract 214: Sumo2 Regulates Oxidative Function Of Adaptor Protein P66shc

SUMOylation is a highly dynamic post-translational modification which regulates protein function and/or stability. It involves conjugation of SUMOs ( S mall U biquitin-Like Mo difier s ) to the lysine residue/s of target proteins mediated via a highly coordinated enzymatic mechanism. Studies have shown that promoting SUMOylation (SUMO2/3) causes vascular endothelial dysfunction and accelerates atherosclerosis. We reported that SUMO2 overexpression in endothelial cells promotes oxidative stress and impairs endothelial function. However, the mechanism is not well understood. Adaptor protein p66Shc is a master regulator of oxidative stress and endothelial function. Genetic deletion of p66Shc protects mice against endothelial dysfunction due to metabolic dysregulation. Prior studies have shown that lysine modification of p66Shc regulates its oxidative function. As SUMOylation primarily occurs on lysine residue, we examined if p66Shc is a target of SUMO2. In this study, we show that p66Shc is a direct target of SUMO2. Using endothelial cells and HEK-293 cells, we show that overexpression of SUMO2 leads to p66Shc SUMO2ylation which is further increased in presence of E2-ligase Ubc9 and reduced by deSUMOylating enzyme SENP1. SUMO2ylation of p66Shc increases oxidative activation (serine-36 phosphorylation) of p66Shc which is essential for reactive oxygen production by p66Shc. Using mass spectroscopic study, we identified that lysine-81 is the only lysine present in CH2 domain which is getting SUMO2ylated. Rendering p66ShcK81 non-sumoylatable (p66ShcK81R) prevented the SUMO2ylation of p66Shc. In endothelial cells, overexpression of p66ShcK81R prevented the SUMO2-induced increase in serine-36 phosphorylation and mitochondrial translocation of p66Shc, which are essential for oxidative function of p66Shc. Collectively, we present a novel molecular mechanism via which SUMO2 regulates the oxidative function of p66Shc and identify a unique post-translational regulation of p66Shc in endothelial cells.

Analysis of Lysine Acetylation and Acetylation-like Acylation In Vitro and In Vivo.

Protein lysine acetylation refers to the covalent transfer of an acetyl moiety from acetyl coenzyme A to the epsilon-amino group of a lysine residue and is critical for regulating protein functions in almost all living cells or organisms. Studies in the past decade have demonstrated the unexpected finding that acetylation-like acylation, such as succinylation, propionylation, butyrylation, crotonylation, and lactylation, is also present in histones and many non-histone proteins. Acetylation and acetylation-like acylation serve as reversible on/off switches for regulating protein function while interplaying with other post-translational modifications (such as phosphorylation and methylation) in a codified manner. Lysine acetylation and acetylation-like acylation are important for regulating different cellular and developmental processes in normal and pathological states. Thus, the detection of such modifications is important for related basic research and molecular diagnostics. Traditionally, lysine acetylation is detected by autoradiography, but recent decades have seen great improvement in the quality of site-specific antibodies against acetylation (or acetylation-like acylation), thereby providing competitive alternatives to the use of radioactive acetate and acetyl-coenzyme A for in vivo and in vitro labeling, respectively. This article describes protocols for the detection of lysine acetylation and acetylation-like acylation with site-specific antibodies to complement extant autoradiography-based methods (Pelletier etal., 2017). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acylation assays in vitro Basic Protocol 2: Determination of in vivo acylation.