scholarly journals Lysine Propionylation is a Widespread Post-Translational Modification Involved in Regulation of Photosynthesis and Metabolism in Cyanobacteria

2019 ◽  
Vol 20 (19) ◽  
pp. 4792 ◽  
Author(s):  
Mingkun Yang ◽  
Hui Huang ◽  
Feng Ge
Keyword(s):  
Large Fraction ◽  
Bioinformatic Analysis ◽  
High Light ◽  
Site Directed Mutagenesis ◽  
Oxygenic Photosynthesis ◽  
Post Translational Modification ◽  
Functional Studies ◽  
Photosynthetic Organisms ◽  
Model Combining ◽  
And Function

Lysine propionylation is a reversible and widely distributed post-translational modification that is known to play a regulatory role in both eukaryotes and prokaryotes. However, the extent and function of lysine propionylation in photosynthetic organisms remains unclear. Cyanobacteria are the most ancient group of Gram-negative bacteria capable of oxygenic photosynthesis, and are of great importance to global carbon and nitrogen cycles. Here, we carried out a systematic study of lysine propionylaiton in cyanobacteria where we used Synechocystis sp. PCC 6803 (Synechocystis) as a model. Combining high-affinity anti-propionyllysine pan antibodies with high-accuracy mass spectrometry (MS) analysis, we identified 111 unique lysine propionylation sites on 69 proteins in Synechocystis. Further bioinformatic analysis showed that a large fraction of the propionylated proteins were involved in photosynthesis and metabolism. The functional significance of lysine propionylation on the enzymatic activity of fructose-1,6-bisphosphatase (FbpI) was studied by site-directed mutagenesis and biochemical studies. Further functional studies revealed that the propionylation level of subunit II of photosystem I (PsaD) was obviously increased after high light (HL) treatment, suggesting that propionylation may be involved in high light adaption in Synechocystis. Thus, our findings provide novel insights into the range of functions regulated by propionylation and reveal that reversible propionylation is a functional modification with the potential to regulate photosynthesis and carbon metabolism in Synechocystis, as well as in other photosynthetic organisms.

scholarly journals Functional identification of potential non-canonical N-glycosylation sites within Cav3.2 T-type calcium channels

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Vendula Ficelova ◽  
Ivana A. Souza ◽  
Leos Cmarko ◽  
Maria A. Gandini ◽  
Robin N. Stringer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Calcium Channels ◽  
Low Voltage ◽  
Consensus Sequence ◽  
Site Directed Mutagenesis ◽  
Post Translational Modification ◽  
Functional Identification ◽  
Electrophysiological Recordings ◽  
Nervous System Function ◽  
And Function

Abstract Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel.

scholarly journals Proteome-wide Analysis of Lysine Acetylation Suggests its Broad Regulatory Scope in Saccharomyces cerevisiae

2012 ◽  
Vol 11 (11) ◽  
pp. 1510-1522 ◽  
Author(s):  
Peter Henriksen ◽  
Sebastian A. Wagner ◽  
Brian T. Weinert ◽  
Satyan Sharma ◽  
Giedrė Bačinskaja ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
High Resolution Mass Spectrometry ◽  
Budding Yeast ◽  
Large Fraction ◽  
Model Organism ◽  
Bioinformatic Analysis ◽  
Lysine Acetylation ◽  
Post Translational Modification ◽  
Acetylation Site

Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with nonacetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites identified several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes.

scholarly journals First comprehensive proteomics analysis of lysine crotonylation in leaves of peanut (Arachis hypogaea L.)

2020 ◽  
Author(s):  
Manlin Xu ◽  
Jianda Luo ◽  
Ying Li ◽  
Lili Shen ◽  
Xia Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Large Scale ◽  
Carbon Fixation ◽  
Post Translational Modification ◽  
Proteomics Analysis ◽  
Photosynthetic Organisms ◽  
Affinity Enrichment ◽  
Arachis Hypogaea L ◽  
Localization Analysis ◽  
And Function

Abstract Background: Lysine crotonylation is an important post-translational modification (PTM) process. Most research in this area has been carried out on mammals and yeast, but there has been no research published on crotonylated proteins on peanut.Results: In the current study, large-scale lysine crotonylation analysis was performed by a combination of affinity enrichment and high-resolution LC-MS/MS analysis. Altogether, 6051 lysine crotonylation sites were identified in 2508 protein groups. Bioinformatics analysis showed that lysine-crotonylated proteins were involved in many biological processes, such as carbon fixation in photosynthetic organisms, photosynthesis, biosynthesis of amino acids, ribosomes, etc. In particular, subcellular localization analysis showed that 43% of the crotonylated proteins were located in the chloroplast and that there were 29 crotonylated proteins associated with photosynthesis. In addition, 26 crotonylated proteins were identified in photosynthesis network and 145 proteins were mapped to ribosome network, indicating the diverse functions for lysine crotonylation in peanut.Conclusion: These data show that crotonylated proteins play a major part in peanut biological functions including carbon fixation in photosynthetic organisms, photosynthesis, biosynthesis of amino acids, ribosomes, etc. A lot of proteins related to photosynthesis and ribosome suggest that lysine crotonylation may play important regulatory roles in their structure and function. This dataset is the first comprehensive proteomics analysis of lysine crotonylation in peanut and will serve as an important resource with which to study the biosynthesis and function of lysine crotonylation in peanut and related plants. Based on these results, further studies to expand on the lysine crotonylation analysis were suggested. Data are available via ProteomeXchange with the identifier PXD017675.

scholarly journals The Sumo proteome of proliferating and neuronal-differentiating cells reveals Utf1 among key Sumo targets involved in neurogenesis

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Juan F. Correa-Vázquez ◽  
Francisco Juárez-Vicente ◽  
Pablo García-Gutiérrez ◽  
Sina V. Barysch ◽  
Frauke Melchior ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Isotope Labeling ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Functional Studies ◽  
Proteomic Approach ◽  
Model Cell ◽  
Negative Effect ◽  
Decapping Enzyme ◽  
And Function

AbstractPost-translational modification by covalent attachment of the Small ubiquitin-like modifier (Sumo) polypeptide regulates a multitude of processes in vertebrates. Despite demonstrated roles of Sumo in the development and function of the nervous system, the identification of key factors displaying a sumoylation-dependent activity during neurogenesis remains elusive. Through a SILAC (stable isotope labeling by/with amino acids in cell culture)-based proteomic approach, we have identified the Sumo proteome of the model cell line P19 under proliferation and neuronal differentiation conditions. More than 300 proteins were identified as putative Sumo targets differentially associated with one or the other condition. A group of proteins of interest were validated and investigated in functional studies. Among these, Utf1 was revealed as a new Sumo target. Gain-of-function experiments demonstrated marked differences between the effects on neurogenesis of overexpressing wild-type and sumoylation mutant versions of the selected proteins. While sumoylation of Prox1, Sall4a, Trim24, and Utf1 was associated with a positive effect on neurogenesis in P19 cells, sumoylation of Kctd15 was associated with a negative effect. Prox1, Sall4a, and Kctd15 were further analyzed in the vertebrate neural tube of living embryos, with similar results. Finally, a detailed analysis of Utf1 showed the sumoylation dependence of Utf1 function in controlling the expression of bivalent genes. Interestingly, this effect seems to rely on two mechanisms: sumoylation modulates binding of Utf1 to the chromatin and mediates recruitment of the messenger RNA-decapping enzyme Dcp1a through a conserved SIM (Sumo-interacting motif). Altogether, our results indicate that the combined sumoylation status of key proteins determines the proper progress of neurogenesis.

scholarly journals Changes in N-Glycosylation Induced By Somatic Hypermutation Modulate the Antigen Reactivity of the Immunoglobulin Receptors in CLL Stereotyped Subset #201

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1733-1733
Author(s):  
Maria Gounari ◽  
Anastasia Iatrou ◽  
Kostantia Kotta ◽  
Ioannis Sarrigeorgiou ◽  
Peggy Lymberi ◽  
...  
Keyword(s):  
Research Funding ◽  
Somatic Hypermutation ◽  
Large Fraction ◽  
Cell Receptor ◽  
Cell Extract ◽  
Lymphocytic Leukemia ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Post Translational Modification ◽  
Antigen Reactivity

The IGHV4-34 gene is intrinsically autoreactive due to carrying a germline(GL)-encoded (super)antigenic motif binding various self (and exogenous) antigens, while it is one of the few IGHV genes that contain a GL-encoded N-glycosylation (N-glyc) site. IGHV4-34 is overrepresented in chronic lymphocytic leukemia (CLL), particularly in cases expressing B cell receptor immunoglobulin (BcR IG) with a significant load of somatic hypermutation (SHM; 'mutated' CLL, M-CLL). Moreover, a large fraction of IGHV4-34 M-CLL cases are clustered in different stereotyped subsets, of which the best studied is subset #4, the largest within M-CLL, defined by the expression of IgG-switched IGHV4-34/IGKV2-30 BcR IG with a distinctive SHM imprint. Considerably smaller than subset #4 is subset #201, defined by the expression of IGHV4-34/IGLV1-44 BcR IG of the IgMD isotype. Subset #201 is noteworthy owing to recurrent replacement SHMs that frequently lead to the creation of novel N- glyc motifs within the VH domain. This may be functionally relevant, considering that N-linked glycosylation is a widespread post-translational modification that is largely SHM-induced during antigen-specific immune responses and can modulate antibody (Ab) affinity towards antigen. That said, nothing is yet known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. In order to obtain insight into this issue, 4 subset #201 clonotypic IGs were expressed as recombinant monoclonal Abs (mAbs) of the mu isotype in HEK293 human cells, in either the authentic SHM state ('wildtype', WT-mAbs) or after reverting specific SHMs that altered N-glyc sites (R-mAbs) by site-directed mutagenesis. Since not all N-glyc motifs are eventually glycosylated, we used the NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for the prediction of N-glycan occupancy. Binding to MEC1 B CLL, Jurkat T and HEK293 cells was assessed by flow cytometry. Reactivity against nuclear Hep-2 cell extract, nDNA, actin, myosin, thyroglobulin (TG), β-amyloid, carbonic anhydrase, F(ab')2 and the non-self hapten trinitrophenyl was tested by ELISA. Non-subset #201 M-CLL mAbs (n=14, including 3 subset #4 mAbs), were used as controls. None of the subset #201 WT-mAbs displayed reactivity in any of the ELISAs. However, unlike most CLL mAbs, all subset #201 WT-mAbs bound to live MEC1 cells, while also exhibiting reactivity to HEK293 cells that was significantly higher when compared to non-subset #201 M-CLL (p=0.0095) or subset #4 (p=0.05); additionally, 1/4 subset #201 mAb displayed weak binding to Jurkat T cells. Three of 4 subset #201 mAbs bore a novel N-glyc site introduced by SHM in codons VL CDR1 36-38 of the clonotypic lambda light chains. Reversion to the GL in one such mAb resulted in enhanced binding to all 3 cell lines [fold change (FC) of binding of the R- vs WT-mAb to MEC1, Jurkat and HEK293: 1.3, 7.9 and 3.3, respectively) and in strong anti-TG activity. The GL-encoded N-glyc site in VH CDR2 57-59, that has been reported to be mostly unoccupied, was targeted by SHM in 2/4 subset #201 mAbs: reversion to GL decreased binding to both MEC1 and HEK293 cells (FC: -8 and -1.4 respectively). Finally, in 2/4 cases, SHM at codons VH FR3 67-68 inserted an N-glyc site that, however, is not predicted to acquire N-glycans. Reversion to GL enhanced the binding of one of these mAbs to MEC1 and HEK293 cells (FC: 2.1 and 5.6, respectively). The same mAb bore an additional predicted N-glyc site introduced by SHM at VH FR3 90-92; reversion of this change to GL augmented binding to both MEC1 and HEK293 cells (FC: 4.1 and 9.7, respectively). Double reversion of both aforementioned SHMs conferred further increased binding than any of the single reversions, implying a synergistic effect. Acquisition of novel N-glyc sites is not an intrinsic characteristic of either M-CLL in general or IGHV4-34 M-CLL in particular and its high incidence in subset #201 implies a selective process likely due to distinct (auto)antigenic pressure. Indeed, subset #201 mAbs exhibit an antigen reactivity profile that differs from that of typical polyreactive mAbs, including natural autoantibodies and other CLL mAbs, binding selectively to viable lymphoblastoid cell line cells and human HEK293 epithelial cells. These results further emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs. Disclosures Chatzidimitriou: Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

scholarly journals Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1393
Author(s):  
Thanyaporn Dechtawewat ◽  
Sittiruk Roytrakul ◽  
Yodying Yingchutrakul ◽  
Sawanya Charoenlappanit ◽  
Bunpote Siridechadilok ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Denv Infection ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modification ◽  
Multifunctional Protein ◽  
Nonstructural Protein 1 ◽  
Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

scholarly journals Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins

2021 ◽  
Vol 22 (5) ◽  
pp. 2647
Author(s):  
M. Quadir Siddiqui ◽  
Maulik D. Badmalia ◽  
Trushar R. Patel
Keyword(s):  
Nucleic Acid ◽  
Nuclear Export ◽  
Consensus Sequence ◽  
Nuclear Export Signal ◽  
Bioinformatic Analysis ◽  
Nucleic Acid Binding ◽  
Protein Protein Interaction ◽  
Lim Domains ◽  
Protein Nucleic Acid ◽  
And Function

Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and functions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share similarities with transcriptional regulators and have positively charged electrostatic patches, which may indicate that they have previously unanticipated nucleic acid binding properties. Intrinsic dynamics analysis of Lim domains suggest that only Lim1 has similar internal dynamics properties, unlike Lim2/3. Furthermore, we analyzed protein expression and mutational frequency in various malignancies, as well as mapped protein-protein interaction networks they are involved in. Overall, our comprehensive bioinformatic analysis suggests that these proteins may play important roles in mediating protein-protein and protein-nucleic acid interactions.

scholarly journals New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Bitam ◽  
Ahmad Elbahnsi ◽  
Geordie Creste ◽  
Iwona Pranke ◽  
Benoit Chevalier ◽  
...  
Keyword(s):  
Phosphinic Acid ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transmembrane Conductance Regulator ◽  
Intracellular Loop ◽  
Respiratory Cells ◽  
Cftr Protein ◽  
Dynamics Simulations ◽  
And Function ◽  
Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

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