Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast

ABSTRACTPolyphosphates (polyP) are long chains of inorganic phosphates that can be attached to lysine residues of target proteins as a non-enzymatic post-translational modification. This modification, termed polyphosphorylation, may be particularly prevalent in bacterial and fungal species that synthesize and store large quantities of polyP. In this study, we applied a proven screening strategy to evaluate the polyphosphorylation status of over 200 candidate targets in the budding yeast S. cerevisiae. We report 8 new polyphosphorylated proteins that interact genetically and physically with a previously identified network of targets implicated in ribosome biogenesis. The expanded target network includes vacuolar proteins Prb1 and Apl5, whose modification with polyP suggests a model for feedback regulation of polyP synthesis, while raising additional questions regarding the location of polyphosphorylation in vivo.

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FPD: A comprehensive phosphorylation database in fungi

10.1101/058867 ◽

2016 ◽

Author(s):

Youhuang Bai ◽

Bin Chen ◽

Yincong Zhou ◽

Silin Ren ◽

Qin Xu ◽

...

Keyword(s):

Critical Role ◽

Fungal Species ◽

Phosphorylation Sites ◽

Post Translational Modification ◽

High Confidence ◽

Cellular Processes ◽

Phosphorylation Motif ◽

Cycle Growth ◽

Available Information

AbstractProtein phosphorylation, one of the most classic post-translational modification, plays a critical role in the diverse cellular processes including cell cycle, growth and signal transduction pathways. However, the available information of phosphorylation in fungi is limited. Here we provided a Fungi Phosphorylation Database (FPD) that comprises high-confidence in vivo phosphosites identified by MS-based proteomics in various fungal species. This comprehensive phosphorylation database contains 62,272 non-redundant phosphorylation sites in 11,222 proteins across eight organisms, including Aspergillus flavus, Aspergillus nidulans, Fusarium graminearum, Magnaporthe oryzae, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Cryptococcus neoformans. A fungi-specific phosphothreonine motif and several conserved phosphorylation motif were discovered by comparatively analyzing the pattern of phosphorylation sites in fungi, plants and animals.Database URL: http://bis.zju.edu.cn/FPD/index.php

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The Hydrogen-Coupled Oligopeptide Membrane Cotransporter Pept2 is SUMOylated in Kidney Distal Convoluted Tubule Cells

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.790606 ◽

2021 ◽

Vol 8 ◽

Author(s):

Takwa S. Aroankins ◽

Sathish K. Murali ◽

Robert A. Fenton ◽

Qi Wu

Keyword(s):

Cellular Response ◽

Apical Membrane ◽

Mouse Kidney ◽

Distal Convoluted Tubule ◽

Wild Type ◽

Post Translational Modification ◽

Physiological Regulation ◽

Lysine Residues ◽

Stimulation Of

Protein post-translational modification by the Small Ubiquitin-like MOdifier (SUMO) on lysine residues is a reversible process highly important for transcription and protein stability. In the kidney, SUMOylation appears to be important for the cellular response to aldosterone. Therefore, in this study, we generated a SUMOylation profile of the aldosterone-sensitive kidney distal convoluted tubule (DCT) as a basis for understanding SUMOylation events in this cell type. Using mass spectrometry-based proteomics, 1037 SUMO1 and 552 SUMO2 sites, corresponding to 546 SUMO1 and 356 SUMO2 proteins, were identified from a modified mouse kidney DCT cell line (mpkDCT). SUMOylation of the renal hydrogen-coupled oligopeptide and drug co-transporter (Pept2) at one site (K139) was found to be highly regulated by aldosterone. Using immunolabelling of mouse kidney sections Pept2 was localized to DCT cells in vivo. Aldosterone stimulation of mpkDCT cell lines expressing wild-type Pept2 or mutant K139R-Pept2, post-transcriptionally increased Pept2 expression up to four-fold. Aldosterone decreased wild-type Pept2 abundance in the apical membrane domain of mpkDCT cells, but this response was absent in K139R-Pept2 expressing cells. In summary, we have generated a SUMOylation landscape of the mouse DCT and determined that SUMOylation plays an important role in the physiological regulation of Pept2 trafficking by aldosterone.

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Vtc5 is localized to the vacuole membrane by the conserved AP-3 complex to regulate polyphosphate synthesis in budding yeast

10.1101/2021.03.15.434853 ◽

2021 ◽

Author(s):

Amanda Bentley-DeSousa ◽

Michael Downey

Keyword(s):

Budding Yeast ◽

Yeast Saccharomyces Cerevisiae ◽

Vacuole Membrane ◽

Dense Granules ◽

Accessory Subunit ◽

High Concentrations ◽

Escrt Complex ◽

Molecular Phenotypes ◽

Inorganic Phosphates

Polyphosphates (polyP) are energy-rich polymers of inorganic phosphates assembled into chains ranging from 3-1000s of residues in length. They are thought to exist in all cells on earth and play roles in an eclectic mix of functions ranging from phosphate homeostasis to cell signaling, infection control, and blood clotting. In the budding yeast Saccharomyces cerevisiae, polyP chains are synthesized by the vacuole-bound VTC complex, which synthesizes polyP while simultaneously translocating it into the vacuole lumen where it is stored at high concentrations. VTC's activity is promoted by an accessory subunit called Vtc5. In this work, we find that the conserved AP-3 complex is required for proper Vtc5 localization to the vacuole membrane. In human cells, previous work has demonstrated that mutation of AP-3 subunits gives rise to Hermansky-Pudlak Syndrome, a rare disease with molecular phenotypes that include decreased polyP accumulation in platelet dense granules. In yeast AP-3 mutants, we find that Vtc5 is rerouted to the vacuole lumen by the ESCRT complex, where it is degraded by the vacuolar protease Pep4. Cells lacking functional AP-3 have decreased levels of polyP, demonstrating that membrane localization of Vtc5 is required for its VTC stimulatory activity in vivo. Our work provides insight into the molecular trafficking of a critical regulator of polyP metabolism in yeast. We speculate that AP-3 may also be responsible for the delivery of polyP regulatory proteins to platelet dense granules in higher eukaryotes.

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Role of Methylglyoxal in Diabetic Cardiovascular and Kidney Diseases: Insights from Basic Science for Application into Clinical Practice

Current Pharmaceutical Design ◽

10.2174/1381612824666180903141832 ◽

2018 ◽

Vol 24 (26) ◽

pp. 3072-3083 ◽

Cited By ~ 6

Author(s):

Sowndramalingam Sankaralingam ◽

Angham Ibrahim ◽

MD Mizanur Rahman ◽

Ali H. Eid ◽

Shankar Munusamy

Keyword(s):

Therapeutic Strategy ◽

Kidney Diseases ◽

Vascular Dysfunction ◽

Dicarbonyl Compound ◽

Renal Complications ◽

Lysine Residues ◽

Patients With Diabetes ◽

Prevalence Of Diabetes Mellitus

Background: The incidence and prevalence of diabetes mellitus are increasing globally at alarming rates. Cardiovascular and renal complications are the major cause of morbidity and mortality in patients with diabetes. Methylglyoxal (MG) - a highly reactive dicarbonyl compound – is increased in patients with diabetes and has been implicated to play a detrimental role in the etiology of cardiovascular and renal complications. Derived from glucose, MG binds to arginine and lysine residues in proteins, and the resultant end products serve as surrogate markers of MG generation in vivo. Under normal conditions, MG is detoxified by the enzyme glyoxalase 1 (Glo1), using reduced glutathione as a co-factor. Elevated levels of MG is known to cause endothelial and vascular dysfunction, oxidative stress and atherosclerosis; all of which are risk factors for cardiovascular diseases. Moreover, MG has also been shown to cause pathologic structural alterations and impair kidney function. Conversely, MG scavengers (such as N-acetylcysteine, aminoguanidine or metformin) or Nrf2/Glo1 activators (such as trans-resveratrol / hesperetin) are shown to be useful in preventing MG-induced cardiovascular and renal complications in diabetes. However, clinical evidence supporting the MG lowering properties of these agents are limited and hence, need further investigation. Conclusion: Reducing MG levels directly using scavengers or indirectly via activation of Nrf2/Glo1 may serve as a novel and potent therapeutic strategy to counter the deleterious effects of MG in diabetic complications.

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Review of Progress in Predicting Protein Methylation Sites

Current Organic Chemistry ◽

10.2174/1385272823666190723141347 ◽

2019 ◽

Vol 23 (15) ◽

pp. 1663-1670 ◽

Cited By ~ 1

Author(s):

Chunyan Ao ◽

Shunshan Jin ◽

Yuan Lin ◽

Quan Zou

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Transcriptional Activity ◽

Regulation Of Gene Expression ◽

Protein Methylation ◽

Biological Processes ◽

Post Translational Modification ◽

Regulatory Enzymes ◽

Lysine Residues ◽

Arginine Residues

Protein methylation is an important and reversible post-translational modification that regulates many biological processes in cells. It occurs mainly on lysine and arginine residues and involves many important biological processes, including transcriptional activity, signal transduction, and the regulation of gene expression. Protein methylation and its regulatory enzymes are related to a variety of human diseases, so improved identification of methylation sites is useful for designing drugs for a variety of related diseases. In this review, we systematically summarize and analyze the tools used for the prediction of protein methylation sites on arginine and lysine residues over the last decade.

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Stu2p binds tubulin and undergoes an open-to-closed conformational change

The Journal of Cell Biology ◽

10.1083/jcb.200511010 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1009-1022 ◽

Cited By ~ 87

Author(s):

Jawdat Al-Bassam ◽

Mark van Breugel ◽

Stephen C. Harrison ◽

Anthony Hyman

Keyword(s):

Conformational Change ◽

Conformational Transition ◽

Budding Yeast ◽

Microtubule Dynamics ◽

Open Structure ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

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N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽

10.3390/molecules26154699 ◽

2021 ◽

Vol 26 (15) ◽

pp. 4699

Author(s):

Mubashir Mintoo ◽

Amritangshu Chakravarty ◽

Ronak Tilvawala

Keyword(s):

Mass Spectrometry ◽

Protease Activity ◽

Viral Infections ◽

Mass Spectrometry Analysis ◽

Post Translational Modification ◽

Spectrometry Analysis ◽

Physiological Conditions ◽

Inflammatory Conditions ◽

Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

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Improvement of Biophysical Properties and Affinity of a Human Anti-L1CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods

International Journal of Molecular Sciences ◽

10.3390/ijms22136696 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6696

Author(s):

Heesu Chae ◽

Seulki Cho ◽

Munsik Jeong ◽

Kiyoung Kwon ◽

Dongwook Choi ◽

...

Keyword(s):

Computational Methods ◽

Human Monoclonal Antibody ◽

Antibody Engineering ◽

Antigen Binding ◽

Post Translational Modification ◽

Biophysical Properties ◽

Preclinical Development ◽

Cell Bank ◽

Aggregation Propensity

The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)—which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue—exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.

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Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications

Proteomes ◽

10.3390/proteomes9020018 ◽

2021 ◽

Vol 9 (2) ◽

pp. 18

Author(s):

Alaa Hseiky ◽

Marion Crespo ◽

Sylvie Kieffer-Jaquinod ◽

François Fenaille ◽

Delphine Pflieger

Keyword(s):

Amino Acid ◽

Small Mass ◽

Mass Region ◽

Amino Acid Sequences ◽

Lysine Modification ◽

Lysine Residues ◽

Two Factors ◽

Low Mass ◽

Diagnostic Ions

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

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ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

Biochemical Journal ◽

10.1042/bj20040590 ◽

2004 ◽

Vol 385 (1) ◽

pp. 309-317 ◽

Cited By ~ 23

Author(s):

Zhefeng ZHAO ◽

Joanna GRUSZCZYNSKA-BIEGALA ◽

Anna ZOLKIEWSKA

Keyword(s):

C2c12 Myotubes ◽

Post Translational Modification ◽

Integrin Β1 ◽

Adp Ribosylation ◽

In Vitro Binding ◽

Vitro Binding ◽

C2c12 Skeletal Muscle Cells ◽

Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

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