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@#Objective To explore the factors affecting the stability of high concentration variable domain of heavy-chain antibody-Fc(VHH-Fc) fusion protein.Methods Three groups of forced degradation experiments,shaking,light and 40℃ high temperature were set up.Differential scanning fluorimetry,dynamic light scattering(DLS) and ultra performance liquid chromatography-mass spectrometry(UPLC-MS) were used to detect the effects of the three forced degradation conditions on the conformational stability,colloidal stability,average hydrodynamic diameter and post-translational modifications of high concentration VHH-Fc fusion protein.Results Under the light condition,the onset temperature of unfolding(T_(onset)),melting temperature(T_m) and aggregation onset temperature(T_(agg)) of high concentration VHH-Fc fusion protein decreased the most,and the oxidation ratio of Met160 and Met266 increased significantly.Under the condition of shaking,the variation of the diffusion interaction parameter(k_D) and the average hydrodynamic diameter was the largest.Conclusion Light can significantly reduce the conformational stability of high concentration VHH-Fc fusion protein and induce methionine oxidation.Shaking has the most significant effect on its colloidal stability and promotes aggregation.
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It is known that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) mediates autophagy through its E3 ubiquitin ligase activity, but the ubiquitinated substrates of SMURF1 need to be further explored. In this paper, the interacting proteins of SMURF1 in THP-1 cells were captured and identified by co-immunoprecipitation (Co-IP) combined with mass spectrometry. It was found that SMURF1 could physically bind to 222 proteins in THP-1 cells, and Adenosine deaminase acting on RNA 1 (ADAR1) had a higher peptide binding score. SMURF1 overexpression vectors were constructed and transfected into HEK-293T cells, then Co-IP and Western blotting assays verified the interaction between exogenous SMURF1 and endogenous ADAR1. qRT-PCR and Western blotting assays were carried out after transfecting SMURF1 overexpression vectors in HEK-293T cells, which identified that overexpression of SMURF1 attenuated the protein levels of ADAR1 (P<0. 05). However, there was no significant difference in the mRNA level of ADAR1. HEK-293T cells with normal and overexpressing SMURF1 were treated with cycloheximide (CHX), respectively, and Western blotting assays showed a shortened half-life of ADAR1 after overexpression of SMURF1 (P < 0. 05). Furthermore, overexpression of SMURF1 increased the polyubiquitination level of ADAR1 as detected by Co-IP and Western blot (P<0. 05). After the proteasome inhibitor (MG132) treatment, the Western blotting assay was performed to demonstrate that the negative regulatory effect of SMURF1 on ADAR1 was weakened after the proteasome degradation pathway was attenuated (P<0. 05). This study shows that SMURF1 interacts with ADAR1, catalyzes the polyubiquitination of ADAR1 and mediates its degradation through the proteasome pathway, which provides a theoretical basis for exploring the various biological functions of SMURF1 by affecting the stability of ADAR1.
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Aim To investigate the potential protein post-translational modifications of psychedelic-induced Head-twith response and underling mechanism. Methods Psychedelics LSD, DOM, or Psilocin was administered to rats by intraperitoneal injection to induce head-twitch response, then the most effective dosage was identified to create animal models of head-twitch behavior. Western blot was performed in detecting the protein phosphorylation, acetylation, and ubiquitination in prefrontal cortex of SD rats after 10 min or 30 min injection. Results LSD (0.025 mg • kg~, i. p.), DOM (3 mg•kg
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Metabolic reprogramming is a hallmark of cancer, including lung cancer. However, the exact underlying mechanism and therapeutic potential are largely unknown. Here we report that protein arginine methyltransferase 6 (PRMT6) is highly expressed in lung cancer and is required for cell metabolism, tumorigenicity, and cisplatin response of lung cancer. PRMT6 regulated the oxidative pentose phosphate pathway (PPP) flux and glycolysis pathway in human lung cancer by increasing the activity of 6-phospho-gluconate dehydrogenase (6PGD) and α-enolase (ENO1). Furthermore, PRMT6 methylated R324 of 6PGD to enhancing its activity; while methylation at R9 and R372 of ENO1 promotes formation of active ENO1 dimers and 2-phosphoglycerate (2-PG) binding to ENO1, respectively. Lastly, targeting PRMT6 blocked the oxidative PPP flux, glycolysis pathway, and tumor growth, as well as enhanced the anti-tumor effects of cisplatin in lung cancer. Together, this study demonstrates that PRMT6 acts as a post-translational modification (PTM) regulator of glucose metabolism, which leads to the pathogenesis of lung cancer. It was proven that the PRMT6-6PGD/ENO1 regulatory axis is an important determinant of carcinogenesis and may become a promising cancer therapeutic strategy.
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Post-translational modification of host proteins induced by pathogenic microorganism plays a critical role in the development, treatment and prevention of diseases. Mycobacterium tuberculosis ( Mtb) is an intracellular pathogen that causes tuberculosis. The post-translational modification induced by Mtb infection is essential in the development and progression of tuberculosis. In recent years, it has been found that Mtb-induced host protein acetylation plays an important role in the regulation of host immunity against tuberculosis, which significantly affects the development of tuberculosis. This review focused on the role and mechanism of Mtb in regulating host protein acetylation, aiming to provide reference for future investigation on potential immunotherapy for tuberculosis.
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Autophagy is a lysosomal-dependent catabolic pathway that is widely present in eukaryote andinvolved in multiple biological functions, such as cytodifferentiation, starvation tolerance and immunedefense. Specially, the autophagy process that recognizes and eliminates intracellular pathogens is definedas xenophagy, which is a vital way for immune cells to execute host defense. However, pathogens haveevolved several strategies to cope with xenophagy via distinct types of virulence factors (effectors, surfaceproteins, etc.). Studies have shown that the autophagy regulatory signals are sophisticated, which areprecisely directed by a variety of autophagy related proteins (ATG proteins). It has been proved that thekey steps of autophagy undergo extensive protein post-translational modifications (PTMs), such asphosphorylation/ dephosphorylation, and ubiquitination/ deubiquitination, etc. These modifications endowthe autophagy regulation with a high degree of dynamics and reversibility via affecting the structure, stability, activity and location of the proteins. Recently, some virulence factors were found to hijackPTMs of the ATG proteins and then affect host autophagy related pathways, thereby resisting xenophagyand promoting pathogens’ survival in the host cell. This review summarizes the current knowledge ofPTMs in xenophagy, especially the mechanisms that pathogens manipulate host xenophagy through PTMs, providing a guidance for exploring xenophagy intervention strategies and controlling infectious diseases.
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Ubiquitination is a unique protein post-translational modification in eukaryotic cells. It regulates a variety of physiological processes, such as protein homeostasis, cell cycle, immune response, DNA repair, and vesicle transport. In view of the importance of ubiquitin in live cells, pathogens have derived a series of effector proteins targeting the host ubiquitin process in the long-term evolutionary process, aiming to regulate the ubiquitin process in the host and render an internal environment suitable for the growth and reproduction of pathogens. Legionella pneumophila is a gram-negative bacterium that is responsible for Legionella pneumoniae pneumonia, causing fever and lung infection with a fatality rate of 15% ~ 30% in severe cases. The Dot / Icm type IV secretory system is the most important virulence system in the infection of Legionella pneumophila. In the process of infecting host cells, Legionella pneumophila uses this secretion system to secrete more than 330 effector proteins to assist bacterial survival, proliferation and escape in host cells. Several effector proteins of Legionella pneumophila regulate the host ubiquitination process directly or indirectly. Recent studies found that some effectors can mediate non-canonical ubiquitination of host proteins, which is different from the classical ubiquitination process. Here, we introduce the latest research progress of novel ubiquitination mediated by effecting proteins of Legionella pneumophila, providing a reference for understanding the important role of ubiquitination in the pathogenesis of Legionella pneumophila.
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During the normal process of biological growth and development, NF-E2-related factor 1 (Nrf1/ Nfe2l1) plays a unique role in maintaining intracellular homeostasis and organ integrity. The deficiency of Nrf1 will lead to severe oxidative stress, genomic instability, and result in liver cancer, neurodegenerative disorder and other diseases. In recent years, it has been found that Nrf1 could yield different activated isoforms or even opposite activity isoforms in a variety of ways to perform distinct functions, and the distribution of these isoforms may play a vital role in the process of tumor development. Therefore, to gain a better understanding of the function of Nrf1 isoforms in cells and tissues, we first briefly introduced its discovery process, and demonstrated the multiple mechanisms of distinct isoform production including selective shear processing, internal selective translation initiation, and post-translation shear processing. More importantly, three different post-translational processing models, transmembrane dynamic processing, site-specific processing, ubiquitin-dependent processing, were expounded in detail. Furthermore, the biological function of different isoforms of Nrf1 and its role in diseases were also summarized in the last section. Collectively, we focus on the production mechanism of different isoforms of Nrf1 and their roles in diseases so as to lay a foundation for finding new strategies for tumor treatment.
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Connexin (Cx), a multigene-encoded transmembrane protein family, forms either gap junctions ( GJ) or hemichannels (HC) to mediate intercellular communication in plasma mem¬brane between adjacent cells or interacts with proteins by its car- boxyl terminal in the cytoplasm to participate in the process of tumor cell proliferation, apoptosis, necrosis, invasion, metasta¬sis, drug resistance and stem cell characteristics.However, mi- slocalization of Cx in cytoplasm or nucleus often occurs in many tumors, and involved in the occurrence and development of tumors.Subcellular localization of Cx is affected by post-transla- tional modifications, including phosphorylation, ubiquitination, and acetylation.In this paper the classification and function of Cx, the relationship between subcellular localization of Cx and tumorigenesis and the regulation of post-translational modifica¬tion on Cx are reviewed in order to provide new ideas for the study of Cx as a potential target for cancer therapy.
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SUMOylation is an important post-translational modification of proteins. Similar to ubiquitylation, SUMOylation is the process that the small ubiquitin-like modifier (SUMO) proteins are specifically and covalently binding to lysine residues of substrate proteins. Through SUMOylation, the physiological functions and pathological processes of cells are well controlled and balanced, and its abnormal activation has been reported in various tumors. Therefore, SUMOylation has been a potential target for anti-tumor drug development. In this review, we summarize recent advances on development of inhibitors targeting SUMOylation pathway and their antitumor properties.
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@#Proteins in the human body are usually made of 20 natural amino acids.Through different amino acid combinations and isomerization, proteins of diverse functions are built.An emerging genetic code expansion technology can introduce unnatural amino acids into specific sites of target protein, endowing the protein with new biological characteristics including covalently binding with proximal proteins, carrying fluorescence, and mimicking specific protein post-translational modifications.In this paper, based on the structure and function of unnatural amino acids, the applications of different types of unnatural amino acids in regulating protein''s stability, studying protein''s conformation, expression level, and localization, and uncovering heretofore unknown protein-protein interactions were reviewed.Besides, genetic code expansion of unnatural amino acids is anticipated to find broad utilities in biomedicine by bringing new ideas and methods to the design and optimization of biologics.
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Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard, Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.
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Lysine acetylation has emerged as one of the most important post-translational modifications that participates in various biological and pathological processes. Histone acetyltransferase 1 (HAT1) as the first identified protein ε-amino lysine acetyltransferase is able to regulate the acetylation of histones and non-histone proteins. However‚ the acetylation substrates and sites mediated by HAT1 in liver cancer are poorly understood. In this study‚ we demonstrated that HAT1 was highly expressed in the liver cancer tissues‚ which was negatively associated with the prognosis of patients. Based on the establishment of the HAT1-knockout HepG2 cell line‚ we employed a quantitative proteomics approach to study the profiling of acetylation mediated by HAT1 in HepG2 cells. Interestingly‚ we identified a total of 858 Kac sites on 547 proteins in the HepG2 cell line‚ in which HAT1 mediated the levels of Kac of 74 sites on 68 proteins. The pathways and metabolic processes that were affected by HAT1-dependent acetylation modification were analyzed by bioinformatics. The results show that Kac regulates disease development‚ RNA biology‚ spliceosome and nucleosome assembly‚ oxidative stress‚ various signaling pathways and metabolic pathways‚ etc.. Moreover‚ we verified that the HAT1-mediated acetylation modification could promote abnormal lipid metabolism. CCK8 assays‚ clone formation and Edu assays revealed that HAT1 could remarkably enhance the cell proliferation of liver cancer in vitro. Thus‚ our finding explored the profiling of HAT1-mediated protein acetylation in HepG2 cells‚ which provides new insights into the underlying mechanism by which HAT1 mediates the development of liver cancer. Clinically‚ the HAT1-mediated acetylation sites could be used for the precise targets of drug development.
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The proteasome is the main complex for specific protein degradation in eukaryotic cells, which plays a key role in protein quality control and cell homeostasis maintenance. It is found that abnormal content or function of the proteasome can lead to many human serious diseases such as cancer and neurodegenerative disorders, and several targeted drugs have been developed for the regulation of proteasome activity. Therefore, it is of great academic value and clinical significance to strengthen the research on the precise regulation mechanism of proteasome activity. The content, assembly and activity of the proteasome are regulated rigorously by multiple levels. In this paper, we summarize the composition subunits, structural features, transcriptional regulation and assembly mechanism of proteasome, and focus on the mechanistic regulation and biological significance of post-translational modifications such as phosphorylation, ubiquitination and acetylation on proteasome, which might be helpful to reveal the regulation mechanism of proteasome in the near future.
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【Objective】 To compare the effects of human albumin products from different domestic manufacturers on human stem cell culture. 【Methods】 Human CD34+ cells were cultured by supplementing human albumin from different manufacturers to serum-free medium, and the expansion ratios of cells within 15 days were counted. Post translational modifications of human albumin products were studied by LC-MS. 【Results】 Supplementing plasma-derived human albumin(pd-alb) to serum-free medium, the expansion ratios of cells could reach up to 20.6±5.7, while the recombinant human albumin(rhAlb) resulted in no significant expansion within 15 days. LC-MS showed significant differences in post-translational modifications from different human albumin products. 【Conclusion】 Different human albumin products showed significantly different effects in the expansion of stem cells due to different sources, processes and stabilizers. Pd-alb products were better for stem cell culture than rhAlb products. There are significant differences between pd-albs and rhAlbs in post-translational modification, but whether these differences are related to stem cell expansion remains to be studied.
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Post-translational modification (PTM) in histone proteins is a covalent modification which mainly consists ofmethylation, phosphorylation, acetylation, ubiquitylation, SUMOylation, glycosylation, and ADP-ribosylation.PTMs have fundamental roles in chromatin structure and function. Histone modifications have also beenknown as epigenetic markers. The PTMs that have taken place in histone proteins can affect gene expressionby altering chromatin structure. Histone modifications act in varied biological processes such as transcriptionalactivation/inactivation, chromosome packaging, mitosis, meiosis, apoptosis, and DNA damage/repair. Defectsin the PTMs pathway have been associated with the occurrence and progression of various human diseases,such as cancer, heart failure, autoimmune diseases, and neurodegenerative disorders such as Parkinson’sdisease, Alzheimer’s disease, and Huntington’s disease. Histone modifications are reversible and used aspotential targets for cancer therapy and prevention. Recent different histone PTMs have key roles in cancercells since it has been shown that histone PTMs markers in cancers are acetylation, methylation, phosphorylation, and ubiquitylation. In this review, we have summarized the six most studied histone modifications andhave examined the role of these modifications in the development of cancer.
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Post-translational modification (PTM) in histone proteins is a covalent modification which mainly consists ofmethylation, phosphorylation, acetylation, ubiquitylation, SUMOylation, glycosylation, and ADP-ribosylation.PTMs have fundamental roles in chromatin structure and function. Histone modifications have also beenknown as epigenetic markers. The PTMs that have taken place in histone proteins can affect gene expressionby altering chromatin structure. Histone modifications act in varied biological processes such as transcriptionalactivation/inactivation, chromosome packaging, mitosis, meiosis, apoptosis, and DNA damage/repair. Defectsin the PTMs pathway have been associated with the occurrence and progression of various human diseases,such as cancer, heart failure, autoimmune diseases, and neurodegenerative disorders such as Parkinson’sdisease, Alzheimer’s disease, and Huntington’s disease. Histone modifications are reversible and used aspotential targets for cancer therapy and prevention. Recent different histone PTMs have key roles in cancercells since it has been shown that histone PTMs markers in cancers are acetylation, methylation, phosphorylation, and ubiquitylation. In this review, we have summarized the six most studied histone modifications andhave examined the role of these modifications in the development of cancer.
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Epigenetic regulation through post-translational modification of histones, especially methylation, is wellconserved in evolution. Although there are several insect genomes sequenced, an analysis with a focus on theirepigenetic repertoire is limited. We have utilized a novel work-flow to identify one or more domains as highpriority domain (HPD), if present in at least 50% of the genes of a given functional class in the referencegenome, namely, that of Drosophila melanogaster. Based on this approach, we have mined histone methyltransferases and demethylases from the whole genome sequence of Aedes aegypti (Diptera), the pea aphidAcyrthosiphon pisum, the triatomid bug Rhodnius prolixus (Hemiptera), the honeybee Apis mellifera (Hymenoptera), the silkworm Bombyx mori (Lepidoptera) and the red flour beetle Tribolium castaneum(Coleoptera). We identified 38 clusters consisting of arginine methyltransferases, lysine methyltransferases anddemethylases using OrthoFinder, and the presence of HPD was queried in these sequences using InterProScan.This approach led us to identify putative novel members and currently inaccurate ones. Other than the highpriority domains, these proteins contain shared and unique domains that can mediate protein–protein interaction. Phylogenetic analysis indicates that there is different extent of protein sequence similarity; averagesimilarity between histone lysine methyltransferases varies from 41% (for active mark) to 48% (for repressivemark), arginine methyltransferases is 51%, and demethylases is 52%. The method utilized here facilitatesreliable identification of desired functional class in newly sequenced genomes
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Proteins exert their roles in life activities via post-translational modifications(PTMs),which include phosphorylation,acetylation,ubiquitination,glycosylation,and methylation.These modifications can change the functions of proteins and play key roles in a variety of diseases.Endometriosis is a common disease in women of childbearing age,although its molecular mechanisms remain unclear.Recent studies have shown that PTMs may be involved in the pathogenesis of endometriosis.Here we review the roles of PTMs in the occurrence and development of endometriosis and the potential medical treatments.
Subject(s)
Female , Humans , Acetylation , Endometriosis/pathology , Glycosylation , Phosphorylation , Protein Processing, Post-Translational , UbiquitinationABSTRACT
Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.