Reversible Post-translational Modification Of Proteins Research Articles

Post-translational modifications in prion diseases.

More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone amino-acids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body's inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrPC), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrPSc) provokes neurodegeneration. PrPC PTCC into PrPSc is an additional type of PTM that affects the tridimensional structure and physiological function of PrPC and generates a protein conformer with neurotoxic properties. PrPC PTCC into PrPSc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt-Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrPC PTCC into PrPSc and stop neurodegeneration in prion diseases. Here, we review known PrPC PTMs that influence PrPC conversion into PrPSc. We summarized how PrPC PTCC into PrPSc impacts the PrPC interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases.

O-GlcNAcylation orchestrates porcine oocyte maturation through maintaining mitochondrial dynamics and function.

O-linked β-N-acetylglucosamine (O-GlcNAc) modification exists widely in cells, playing a crucial role in the regulation of important biological processes such as transcription, translation, metabolism, and the cell cycle. O-GlcNAc modification is an inducible reversible dynamic protein post-translational modification, which regulates complex cellular activities through transient glycosylation and deglycosylation. O-GlcNAc glycosylation is specifically regulated by O-GlcNAc glycosyltransferase (O-GlcNAc transferase, OGT) and O-GlcNAc glycoside hydrolase (O-GlcNAcase). However, the mechanisms underlying the effects of O-GlcNAc modification on the female reproductive system, especially oocyte quality, remain unclear. Here, we found that after OGT was inhibited, porcine oocytes failed to extrude the first polar body and exhibited abnormal actin and microtubule assembly. Meanwhile, the mitochondrial dynamics and function were also disrupted after inhibition of OGT function, resulting in the occurrence of oxidative stress and autophagy. Collectively, these results inform our understanding of the importance of the glycosylation process for oocyte maturation, especially for the maturation quality of porcine oocytes, and the alteration of O-GlcNAc in oocytes to regulate cellular events deserves further investigation.

Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains

Ubiquitination is a reversible protein post-translational modification in which consequent enzymatic activity results in the covalent linking of ubiquitin to a target protein. Once ubiquitinated, a protein can undergo multiple rounds of ubiquitination on multiple sites or form poly-ubiquitin chains. Ubiquitination regulates various cellular processes, and dysregulation of ubiquitination has been associated with more than one type of cancer. Therefore, efforts have been carried out to identify modulators of the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor ubiquitination activity of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a good signal window with a robust average Z’ factor of 0.76 on 384-well microplates, indicating a good assay for screening inhibitors in a high-throughput setting. From a validatory screening experiment, we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. We also demonstrate that the method can be expanded to study deubiquitinases, such as USP28, that reduce FRET due to hydrolysis of fluorescent poly-ubiquitin chains.

Competition for cysteine acylation by C16:0 and C18:0 derived lipids is a global phenomenon in the proteome

S-acylation is a reversible posttranslational protein modification consisting of attachment of a fatty acid to a cysteine via a thioester bond. Research over the last few years has shown that a variety of different fatty acids, such as palmitic acid (C16:0), stearate (C18:0), or oleate (C18:1), are used in cells to S-acylate proteins. We recently showed that GNAI proteins can be acylated on a single residue, Cys3, with either C16:0 or C18:1, and that the relative proportion of acylation with these fatty acids depends on the level of the respective fatty acid in the cell's environment. This has functional consequences for GNAI proteins, with the identity of the acylating fatty acid affecting the subcellular localization of GNAIs. Unclear is whether this competitive acylation is specific to GNAI proteins or a more general phenomenon in the proteome. We perform here a proteome screen to identify proteins acylated with different fatty acids. We identify 218 proteins acylated with C16:0 and 308 proteins acylated with C18-lipids, thereby uncovering novel targets of acylation. We find that most proteins that can be acylated by C16:0 can also be acylated with C18-fatty acids. For proteins with more than one acylation site, we find that this competitive acylation occurs on each individual cysteine residue. This raises the possibility that the function of many different proteins can be regulated by the lipid environment via differential S-acylation.

Roles of ADP-Ribosylation during Infection Establishment by Trypanosomatidae Parasites.

ADP-ribosylation is a reversible post-translational protein modification, which is evolutionarily conserved in prokaryotic and eukaryotic organisms. It governs critical cellular functions, including, but not limited to cellular proliferation, differentiation, RNA translation, and genomic repair. The addition of one or multiple ADP-ribose moieties can be catalysed by poly(ADP-ribose) polymerase (PARP) enzymes, while in eukaryotic organisms, ADP-ribosylation can be reversed through the action of specific enzymes capable of ADP-ribose signalling regulation. In several lower eukaryotic organisms, including Trypanosomatidae parasites, ADP-ribosylation is thought to be important for infection establishment. Trypanosomatidae encompasses several human disease-causing pathogens, including Trypanosoma cruzi, T. brucei, and the Leishmania genus. These parasites are the etiological agents of Chagas disease, African trypanosomiasis (sleeping sickness), and leishmaniasis, respectively. Currently, licenced medications for these infections are outdated and often result in harmful side effects, and can be inaccessible to those carrying infections, due to them being classified as neglected tropical diseases (NTDs), meaning that many infected individuals will belong to already marginalised communities in countries already facing socioeconomic challenges. Consequently, funding to develop novel therapeutics for these infections is overlooked. As such, understanding the molecular mechanisms of infection, and how ADP-ribosylation facilitates infection establishment by these organisms may allow the identification of potential molecular interventions that would disrupt infection. In contrast to the complex ADP-ribosylation pathways in eukaryotes, the process of Trypanosomatidae is more linear, with the parasites only expressing one PARP enzyme, compared to the, at least, 17 genes that encode human PARP enzymes. If this simplified pathway can be understood and exploited, it may reveal new avenues for combatting Trypanosomatidae infection. This review will focus on the current state of knowledge on the importance of ADP-ribosylation in Trypanosomatidae during infection establishment in human hosts, and the potential therapeutic options that disrupting ADP-ribosylation may offer to combat Trypanosomatidae.

Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.

Abstract LB318: SB-4826, a first-in-class oral, covalent inhibitor of SUMO E1 that induces IFN signaling and inhibits tumor growth as monotherapy and in combination with immune checkpoint blockade

Abstract Sumoylation is a reversible post-translational modification of proteins by small ubiquitin-like modifier (SUMO) that regulates protein function and contributes to cellular response to stress. Sumoylation is an essential pathway for all cells and is involved in diverse processes such as inflammation, DNA damage response, signaling and cell survival/apoptosis. Due to its involvement in such cellular functions, sumoylation intersects with the majority of the hallmarks of cancer. Many cancers display elevated expression of SUMO pathway components, a mechanism co-opted by tumors to survive under stressful conditions. In addition to its tumor intrinsic functions, sumoylation has emerged as a novel target for activating anti-tumor immunity due to its role in regulating type I interferon (IFN) signaling. Targeting sumoylation is a compelling anti-cancer strategy due to the multiple mechanisms of action that may drive anti-tumor activity for cancer patients. We report the discovery of SB-4826, a novel, orally active, covalent small molecule inhibitor of the Sumo Activating Enzyme (SUMO E1), the initiating enzyme of the sumoylation cascade. SB-4826 selectively and irreversibly binds an allosteric pocket of SUMO E1 and drives potent biochemical and cellular activity. SB-4826 inhibits global sumoylation in cells and blocks proliferation across a broad panel (100+) of cancer cell lines. Cysteine-proteome profiling by mass spectrometry indicated that Cys-30 of SUMO E1 was the only cysteine-containing peptide (out of 14,541 measured) that met the criteria for covalent engagement indicating exquisite selectivity across the cellular proteome. In an in vivo pharmacodynamic model measuring IFN signaling SB-4826 treatment led to dose- and time-dependent induction of IFN regulated cytokines. SB-4826 treatment resulted in significant tumor growth inhibition in multiple in vivo models. In a syngeneic A20 lymphoma model SB-4826 led to tumor stasis. In a human RAJI xenograft model, SB-4826 induced near-complete inhibition of tumor growth as a single agent and resulted in tumor regression when combined with rituximab. In a mouse CT-26 colorectal tumor model which is generally refractory to anti-PD-1 treatment, SB-4826 significantly inhibited tumor growth as monotherapy and led to 60% complete tumor responses when combined with anti-PD-1. SB-4826 caused increased CD8+ T cell infiltration in CT-26 tumors supporting the role of SB-4826 in inducing IFN signaling and leading to a tumor microenvironment that is more responsive to immune checkpoint inhibition. SUMO E1 is a clinically validated cancer target as evidenced by recent data showing encouraging responses to TAK-981, an IV-administered, ATP-competitive inhibitor. SB-4826 is a differentiated and optimized SUMO E1 inhibitor that is administered orally and demonstrated improved activity in preclinical models. SB-4826 has favorable properties for development, is a new and promising cancer therapy, and is entering phase 1 clinical studies in 2023. Citation Format: Jude R. Canon, Ashley Callinan, Sarah Bradley, Laurie Wang, Mackenzie Kui, Ariel Wein, Gabriele Sulli, Peter Buchowiecki, Fang-Tsao Hong, Miles Kubota, Mary Walton, Victor J. Cee. SB-4826, a first-in-class oral, covalent inhibitor of SUMO E1 that induces IFN signaling and inhibits tumor growth as monotherapy and in combination with immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB318.

Studying Reversible Protein Post-translational Modification through Co-translational Modification.

Understanding the post-translational modifications of targeted proteins is of great significance for manipulating the physiological processes of eukaryotes. Chemical biology tools have been used to investigate the biological roles of those post-translational modifications at particular sites, especially genetic code expansion technology, which can also be combined with the concept of synthetic biology to generate a genetically modified organism with a synthetic auxotroph for co-translational modification components. In this concept, we will introduce applications, limitations, and perspectives of genetic code expansion technology for studying post-translational modification based on recent progresses. Future perspectives of genetically modified organisms also will be discussed in regard to the application of post-translational modification research.

Acetylation of nuclear receptors in health and disease: an update.

Lysine acetylation is a common reversible post-translational modification of proteins that plays a key role in regulating gene expression. Nuclear receptors (NRs) include ligand-inducible transcription factors and orphan receptors for which the ligand is undetermined, which together regulate the expression of genes involved in development, metabolism, homeostasis, reproduction and human diseases including cancer. Since the original finding that the ERα, AR and HNF4 are acetylated, we now understand that the vast majority of NRs are acetylated and that this modification has profound effects on NR function. Acetylation sites are often conserved and involve both ordered and disordered regions of NRs. The acetylated residues function as part of an intramolecular signalling platform intersecting phosphorylation, methylation and other modifications. Acetylation of NR has been shown to impact recruitment into chromatin, co-repressor and coactivator complex formation, sensitivity and specificity of regulation by ligand and ligand antagonists, DNA binding, subcellular distribution and transcriptional activity. A growing body of evidence in mice indicates a vital role for NR acetylation in metabolism. Additionally, mutations of the NR acetylation site occur in human disease. This review focuses on the role of NR acetylation in coordinating signalling in normal physiology and disease.

Lysine acetylome profiling in mouse hippocampus and its alterations upon FMRP deficiency linked to abnormal energy metabolism

Loss of fragile X retardation protein (FMRP) leads to fragile X syndrome (FXS), a common cause of inherited intellectual disability. Protein lysine acetylation (K-ac), a reversible post-translational modification of proteins, is associated with the regulation of brain development and neuropathies. However, a comprehensive hippocampal K-ac protein profile in response to FMRP deficiency has not been reported until now. Using LC-MS/MS to analyze the enriched K-ac peptides, this study identified 1629 K-ac hits across 717 proteins in the mouse hippocampus, and these proteins were enriched in several metabolic processes. Of them, 51 K-ac hits across 45 proteins were significantly changed upon loss of FMRP. These altered K-ac proteins were enriched in energy metabolic processes including carboxylic acid metabolism process, aerobic respiration and citrate cycle, linking with several neurological disorders such as lactic acidosis, Lewy body disease, Leigh disease and encephalopathies. In the mouse hippocampus and the hippocampal HT-22 cells, FMRP deficiency could induce altered K-ac modification of several key enzymes, decrease in ATP and increase in lactate. Thus, this study identified a global hippocampal lysine acetylome and an altered K-ac protein profile upon loss of FMRP linked to abnormal energy metabolism, implicating in the pathogenesis of FXS. SignificanceFragile X syndrome (FXS) is a common inherited neurodevelopment disorder characterized by intellectual disability and an increased risk for autism spectrum disorder. FXS is resulted from silencing of the FMR1 gene, which induces loss of its encoding protein FMRP. Molecular and metabolic changes of Fmr1-null animal models of FXS have been identified to potentially contribute to the pathogenesis of FXS. Here, we used a TMT-labeled quantitative proteomic analysis of the peptides enriched by anti-K-ac antibodies and identified a global K-ac protein profile in the mouse hippocampus with a total of 1629 K-ac peptides on 717 proteins. Of them, 51 K-ac peptides regarding 45 proteins altered in response to loss of FMRP, which were enriched in energy metabolic processes and were implicated in several neurological disorders. Thus this study for the first time provides a global hippocampal lysine acetylome upon FMRP deficiency linked to abnormal metabolic pathways, which may contribute to pathogenic mechanism of FXS.

Dynamic switching of crotonylation to ubiquitination of H2A at lysine 119 attenuates transcription-replication conflicts caused by replication stress.

The reversible post-translational modification (PTM) of proteins plays an important role in many cellular processes. Lysine crotonylation (Kcr) is a newly identified PTM, but its functional significance remains unclear. Here, we found that Kcr is involved in the replication stress response. We show that crotonylation of histone H2A at lysine 119 (H2AK119) and ubiquitination of H2AK119 are reversibly regulated by replication stress. Decrotonylation of H2AK119 by SIRT1 is a prerequisite for subsequent ubiquitination of H2AK119 by BMI1. Accumulation of ubiquitinated H2AK119 at reversed replication forks leads to the release of RNA Polymerase II and transcription repression in the vicinity of stalled replication forks. These effects attenuate transcription–replication conflicts (TRCs) and TRC-associated R-loop formation and DNA double-strand breaks. These findings suggest that decrotonylation and ubiquitination of H2A at lysine 119 act together to resolve replication stress-induced TRCs and protect genome stability.

Abstract P1081: Palmitoylation-dependent Regulation Of Rab3gap1, Cardiomyocyte Exocytosis, And Atrial Natriuretic Peptide Secretion

Palmitoylation is the reversible post-translational modification of proteins with fatty acids catalyzed by zDHHC S-acyltransferases that functions as a potent regulatory mechanism controlling protein trafficking and signal transduction. To investigate functions of palmitoylation in cardiac homeostasis and pathophysiology, we generated transgenic mice with cardiomyocyte-specific overexpression of the Golgi enzyme, zDHHC9, which was previously reported as the Ras S-acyltransferase and is associated with X-linked intellectual disability. Although we did not observe altered Ras palmitoylation, zDHHC9 transgenic mice develop age-dependent cardiac hypertrophy that progresses to functional decompensation and cardiomyopathy. We found palmitoylation of Rab3 GTPase activating protein 1 (Rab3gap1) was significantly elevated in zDHHC9 overexpressing hearts prior to disease onset concomitant with enhanced GTP-loading of Rab3a, suggesting zDHHC9-mediated palmitoylation impairs Rab3gap1-dependent nucleotide cycling on Rab3a. Consistent with this, zDHHC9 overexpression resulted in enhanced Rab3gap1 retention at the Golgi and expansion of Rab3a-positive vesicles at the cardiomyocyte periphery. Co-immunoprecipitation analysis in HEK293AD cells confirmed that zDHHC9 elevation diminishes interaction between active Rab3a and Rab3gap1. To interrogate roles of zDHHC9 in cardiomyocyte exocytosis, we assessed secretion of atrial natriuretic peptide (ANP) and surprisingly found that while zDHHC9 overexpression increased intracellular retention and reduced release of ANP, knockdown of zDHHC9 promoted phenylephrine-induced ANP secretion. Taken together, these data suggest zDHHC9-mediated palmitoylation of Rab3gap1 represses ANP secretion by disrupting GTP:GDP cycling on Rab3a necessary for release of ANP from secretory granules. These studies reveal unexpected functions for zDHHC9-mediated palmitoylation in modulation of secretory pathway flux and regulated hormone secretion via downstream impacts on Rab3a-mediated vesicle fusion and exocytosis and suggest novel roles for zDHHC9, Rab3gap1, and Rab3a in natriuretic peptide secretion.

Neddylation tunes peripheral blood mononuclear cells immune response in COVID-19 patients

The COVID-19 pandemic caused by SARS-CoV-2 has reached 5.5 million deaths worldwide, generating a huge impact globally. This highly contagious viral infection produces a severe acute respiratory syndrome that includes cough, mucus, fever and pneumonia. Likewise, many hospitalized patients develop severe pneumonia associated with acute respiratory distress syndrome (ARDS), along an exacerbated and uncontrolled systemic inflammation that in some cases induces a fatal cytokine storm. Although vaccines clearly have had a beneficial effect, there is still a high percentage of unprotected patients that develop the pathology, due to an ineffective immune response. Therefore, a thorough understanding of the modulatory mechanisms that regulate the response to SARS-CoV-2 is crucial to find effective therapeutic alternatives. Previous studies describe the relevance of Neddylation in the activation of the immune system and its implications in viral infection. In this context, the present study postulates Neddylation, a reversible ubiquitin-like post-translational modification of proteins that control their stability, localization and activity, as a key regulator in the immune response against SARS-CoV-2. For the first time, we describe an increase in global neddylation levels in COVID-19 in the serum of patients, which is particularly associated with the early response to infection. In addition, the results showed that overactivation of neddylation controls activation, proliferation, and response of peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 patients. Inhibition of neddylation, and the subsequent avoidance of activated PBMCs, reduces cytokine production, mainly IL-6 and MCP-1 and induce proteome modulation, being a critical mechanism and a potential approach to immunomodulate COVID-19 patients.

Panoramic Perspective on Human Phosphosites.

Protein phosphorylation is the most common reversible post-translational modification of proteins and is key in the regulation of many cellular processes. Due to this importance, phosphorylation is extensively studied, resulting in the availability of a large amount of mass spectrometry-based phospho-proteomics data. Here, we leverage the information in these large-scale phospho-proteomics data sets, as contained in Scop3P, to analyze and characterize proteome-wide protein phosphorylation sites (P-sites). First, we set out to differentiate correctly observed P-sites from false-positive sites using five complementary site properties. We then describe the context of these P-sites in terms of the protein structure, solvent accessibility, structural transitions and disorder, and biophysical properties. We also investigate the relative prevalence of disease-linked mutations on and around P-sites. Moreover, we assess the structural dynamics of P-sites in their phosphorylated and unphosphorylated states. As a result, we show how large-scale reprocessing of available proteomics experiments can enable a more reliable view on proteome-wide P-sites. Furthermore, adding the structural context of proteins around P-sites helps uncover possible conformational switches upon phosphorylation. Moreover, by placing sites in different biophysical contexts, we show the differential preference in protein dynamics at phosphorylated sites when compared to the nonphosphorylated counterparts.

Redox Regulation of Soluble Epoxide Hydrolase—Implications for Cardiovascular Health and Disease

Cell responses to changes in their redox state are significantly mediated by reversible oxido-reductive post-translational modifications of proteins, potentially altering their activities or interactions. These modifications are important for the homeostatic responses of cells to environmental changes that alter their redox state. Such redox regulatory mechanisms not only operate to maintain health, but can become dysregulated and contribute to pathophysiology. In this review, we focus on the redox control of soluble epoxide hydrolase (sEH), which is widely expressed, including in blood vessels and cardiomyocytes. We review the different types of oxidative modifications that regulate sEH and how they may alter cardiovascular physiology and affect disease progression during stress.

A GFP-based ratiometric sensor for cellular methionine oxidation.

Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation. Placed at amino-acid position 147 in an otherwise methionine-less sfGFP, the oxidation of this specific methionine to methionine sulfoxide results in a ratiometric fluorescence change when excited with ∼400 and ∼470nm light. The strength and homogeneity of the sensor expression is suited for live-cell imaging as well as fluorescence-activated cell sorting (FACS) experiments using standard laser wavelengths (405/488nm). Expressed in mammalian cells and also in S. cerevisiae, the sensor protein faithfully reports on the status of methionine oxidation in an integrating manner. Variants targeted to membranes and the mitochondria provide subcellular resolution of methionine oxidation, e.g. reporting on site-specific oxidation by illumination of endogenous protoporphyrin IX.

Tip growth defective1 interacts with cellulose synthase A3 to regulate cellulose biosynthesis in Arabidopsis.

AtTIP1 physically and genetically interacts with AtCESA3. AtCESA3 undergoes S-acylation, possibly mediated by AtTIP1, suggesting a specific role of AtTIP1 in cellulose biosynthesis and plant development. S-acylation is a reversible post-translational lipid modification of proteins catalyzed by protein S-acyl transferases (PATs). S-acylation is important for various biological molecular mechanisms including cellulose biosynthesis. Cellulose is synthesized by the cellulose synthase A (CESA) complexes (CSCs) at the plasma membrane. However, specific PAT involving in cellulose biosynthesis has not been identified and the precise mechanism by which PAT regulates the CESAs is largely unknown. Here, we report isolation of tip1-5, an allele of Tip Growth Defective1 (AtTIP1/AtPAT24) with a premature stop codon. tip1-5 genetically interacts with ixr1-2, a point mutant of AtCESA3 which encodes a catalytic subunit of CSC synthesizing primary wall cellulose. We show that AtTIP1 physically interacts with AtCESA3. AtCESA3 undergoes S-acylation, which is possibly mediated by AtTIP1, suggesting a functional relationship between AtTIP1 and AtCESA3. Moreover, the interfascicular fiber cells in the primary inflorescence stems of tip1-5 ixr1-2 double mutant contain thinner cell walls and significantly less crystalline cellulose compared to the single mutants. These results highlight the positive regulation of AtTIP1 in cellulose biosynthesis, and a specific role of AtPAT in plant development.

Facile purification of active recombinant mouse cytosolic carboxypeptidase 6 from Escherichia coli

CCP6 is a member of cytosolic carboxypeptidases (CCPs) family, an eraser of a reversible protein posttranslational modification — polyglutamylation, and represents a potential therapeutic target. Currently, production of CCPs mainly depends on eukaryotic expression system, which is time-consuming and costly. Here, we reported that mouse origin full-length CCP6 can be successfully expressed in the soluble fraction of bacteria ArcticExpress (DE3) strain. However, the recombinant mCCP6 was initially co-purified with Cpn60 in a stoichiometric ratio of roughly 1:7 and exhibited no enzyme activity. When coupled with a step to promote the release of the substrate protein from the chaperonins by treatment with ATP/Mg2+/K+, the recombinant CCP6 with deglutamylation activity was obtained, though still partially associated with Cpn60. This is the first report, to our knowledge, that the successful expression and purification of active recombinant mammalian CCPs using a bacterial system was achieved.

Dual-Role Ubiquitination Regulation Shuttling the Entire Life Cycle of the Flaviviridae.

Ubiquitination is a reversible protein post-translational modification that regulates various pivotal physiological and pathological processes in all eukaryotes. Recently, the antiviral immune response is enhanced by the regulation of ubiquitination. Intriguingly, Flaviviridae viruses can ingeniously hijack the ubiquitination system to help them survive, which has become a hot topic among worldwide researchers. The Flaviviridae family members, such as HCV and CSFV, can cause serious diseases of humans and animals around the world. The multiple roles of ubiquitination involved in the life cycle of Flaviviridae family would open new sight for future development of antiviral tactic. Here, we discuss recent advances with regard to functional roles of ubiquitination and some ubiquitin-like modifications in the life cycle of Flaviviridae infection, shedding new light on the antiviral mechanism research and therapeutic drug development.

Recent Development of Machine Learning Methods in Sumoylation Sites Prediction.

Sumoylation of proteins is an important reversible post-translational modification of proteins and mediates a variety of cellular processes. Sumo-modified proteins can change their subcellular localization, activity, and stability. In addition, it also plays an important role in various cellular processes such as transcriptional regulation and signal transduction. The abnormal sumoylation is involved in many diseases, including neurodegeneration and immune-related diseases, as well as the development of cancer. Therefore, identification of the sumoylation site (SUMO site) is fundamental to understanding their molecular mechanisms and regulatory roles. In contrast to labor-intensive and costly experimental approaches, computational prediction of sumoylation sites in silico has also attracted much attention for its accuracy, convenience, and speed. At present, many computational prediction models have been used to identify SUMO sites, but their contents have not been comprehensively summarized and reviewed. Therefore, the research progress of relevant models is summarized and discussed in this paper. We have briefly summarized the development of bioinformatics methods for sumoylation site prediction by mainly focusing on the benchmark dataset construction, feature extraction, machine learning method, published results, and online tools. We hope that this review will provide more help for wet-experimental scholars.