scholarly journals Quantitative proteomics reveals extensive lysine ubiquitination in the Arabidopsis root proteome and uncovers novel transcription factor stability states

2021 ◽  
Author(s):  
Gaoyuan Song ◽  
Damilola Olatunji ◽  
Christian Montes ◽  
Natalie M Clark ◽  
Yunting Pu ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Site Directed Mutagenesis ◽  
Biological Processes ◽  
Protein Activity ◽  
Helix Loop Helix ◽  
Primary Roots ◽  
Lysine Residues ◽  
Ubiquitin Proteasome ◽  
The Stability

Protein activity, abundance, and stability can be regulated by posttranslational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function and yet we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich peptides containing the di-glycine (diGly) remnant of ubiquitin and coupled that with isobaric labeling to enable quantification, from up to 16-multiplexed samples, for plant tissues. Collectively, we identified 7,130 diGly-modified lysine residues sites arising from 3,178 proteins in Arabidopsis primary roots. These data include ubiquitin proteasome dependent ubiquitination events as well as ubiquitination events associated with auxin treatment. Gene Ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and root morphogenesis. We determined the ubiquitinated lysine residues that directly regulate the stability of the transcription factors CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) using site directed mutagenesis and in vivo degradation assays. These comprehensive site-level ubiquitinome profiles provide a wealth of data for future studies related to modulation of biological processes mediated by this posttranslational modification in plants.

scholarly journals SCL Assembles a Multifactorial Complex That Determines Glycophorin A Expression

2004 ◽  
Vol 24 (4) ◽  
pp. 1439-1452 ◽  
Author(s):  
Rachid Lahlil ◽  
Eric Lécuyer ◽  
Sabine Herblot ◽  
Trang Hoang
Keyword(s):  
Transcription Factors ◽  
Hematopoietic Cells ◽  
Inhibitory Effect ◽  
Erythroid Cell ◽  
Glycophorin A ◽  
Protein Activity ◽  
Protein Levels ◽  
Helix Loop Helix

ABSTRACT SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.

scholarly journals Targeting KDM4A-AS1 represses AR/AR-Vs deubiquitination and enhances enzalutamide response in CRPC

Oncogene ◽  
2021 ◽  
Author(s):  
Boya Zhang ◽  
Mingpeng Zhang ◽  
Yanjie Yang ◽  
Qi Li ◽  
Jianpeng Yu ◽  
...  
Keyword(s):  
Splice Variants ◽  
Tumor Promoter ◽  
Castration Resistant Prostate Cancer ◽  
Castration Resistant ◽  
Ubiquitin Proteasome ◽  
Application Potential ◽  
The Stability ◽  
Cancer Tissues

AbstractCastration-resistant prostate cancer (CRPC) is a highly malignant type of advanced cancer resistant to androgen deprivation therapy. One of the important mechanisms for the development of CRPC is the persistent imbalanced regulation of AR and AR splice variants (AR/AR-Vs). In this study, we reported KDM4A-AS1, a recently discovered lncRNA, as a tumor promoter that was significantly increased in CRPC cell lines and cancer tissues. Depletion of KDM4A-AS1 significantly reduced cell viability, proliferation, migration in vitro, and tumor growth in vivo. We found that by binding to the NTD domain, KDM4A-AS1 enhances the stability of USP14-AR/AR-Vs complex, and promoted AR/AR-Vs deubiquitination to protect it from MDM2-mediated ubiquitin-proteasome degradation. Moreover, KDM4A-AS1 was found to enhance CRPC drug resistance to enzalutamide by repressing AR/AR-Vs degradation; antisense oligonucleotide drugs targeting KDM4A-AS1 significantly reduced the growth of tumors with enzalutamide resistance. Taken together, our results indicated that KDM4A-AS1 played an important role in the progression of CRPC and enzalutamide resistance by regulating AR/AR-Vs deubiquitination; targeting KDM4A-AS1 has broad clinical application potential.

scholarly journals SUMO: From Bench to Bedside

2020 ◽  
Vol 100 (4) ◽  
pp. 1599-1619 ◽  
Author(s):  
Hui-Ming Chang ◽  
Edward T. H. Yeh
Keyword(s):  
Cellular Localization ◽  
Protein Modification ◽  
Redox Regulation ◽  
Biological Processes ◽  
Protein Activity ◽  
Protein Protein Interaction ◽  
The Family ◽  
Lysine Residues ◽  
Sumo Pathway ◽  
Posttranslational Protein Modification

Sentrin/small ubiquitin-like modifier (SUMO) is protein modification pathway that regulates multiple biological processes, including cell division, DNA replication/repair, signal transduction, and cellular metabolism. In this review, we will focus on recent advances in the mechanisms of disease pathogenesis, such as cancer, diabetes, seizure, and heart failure, which have been linked to the SUMO pathway. SUMO is conjugated to lysine residues in target proteins through an isopeptide linkage catalyzed by SUMO-specific activating (E1), conjugating (E2), and ligating (E3) enzymes. In steady state, the quantity of SUMO-modified substrates is usually a small fraction of unmodified substrates due to the deconjugation activity of the family Sentrin/SUMO-specific proteases (SENPs). In contrast to the complexity of the ubiquitination/deubiquitination machinery, the biochemistry of SUMOylation and de-SUMOylation is relatively modest. Specificity of the SUMO pathway is achieved through redox regulation, acetylation, phosphorylation, or other posttranslational protein modification of the SUMOylation and de-SUMOylation enzymes. There are three major SUMOs. SUMO-1 usually modifies a substrate as a monomer; however, SUMO-2/3 can form poly-SUMO chains. The monomeric SUMO-1 or poly-SUMO chains can interact with other proteins through SUMO-interactive motif (SIM). Thus SUMO modification provides a platform to enhance protein-protein interaction. The consequence of SUMOylation includes changes in cellular localization, protein activity, or protein stability. Furthermore, SUMO may join force with ubiquitin to degrade proteins through SUMO-targeted ubiquitin ligases (STUbL). After 20 yr of research, SUMO has been shown to play critical roles in most, if not all, biological pathways. Thus the SUMO enzymes could be targets for drug development to treat human diseases.

scholarly journals Protein S-palmitoylation in cellular differentiation

2017 ◽  
Vol 45 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Mingzi M. Zhang ◽  
Howard C. Hang
Keyword(s):  
Posttranslational Modification ◽  
Protein Function ◽  
Cellular Differentiation ◽  
Protein S ◽  
Biological Processes ◽  
Global Studies ◽  
Technological Advances ◽  
Protein Palmitoylation ◽  
Spatiotemporal Control

Reversible protein S-palmitoylation confers spatiotemporal control of protein function by modulating protein stability, trafficking and activity, as well as protein–protein and membrane–protein associations. Enabled by technological advances, global studies revealed S-palmitoylation to be an important and pervasive posttranslational modification in eukaryotes with the potential to coordinate diverse biological processes as cells transition from one state to another. Here, we review the strategies and tools to analyze in vivo protein palmitoylation and interrogate the functions of the enzymes that put on and take off palmitate from proteins. We also highlight palmitoyl proteins and palmitoylation-related enzymes that are associated with cellular differentiation and/or tissue development in yeasts, protozoa, mammals, plants and other model eukaryotes.

scholarly journals The Molecular Basis of Ubiquitin-Conjugating Enzymes (E2s) as a Potential Target for Cancer Therapy

2021 ◽  
Vol 22 (7) ◽  
pp. 3440
Author(s):  
Xiaodi Du ◽  
Hongyu Song ◽  
Nengxing Shen ◽  
Ruiqi Hua ◽  
Guangyou Yang
Keyword(s):  
Therapeutic Target ◽  
Molecular Basis ◽  
Biological Processes ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Conjugating Enzymes ◽  
The Stability ◽  
Ubiquitin Conjugating Enzymes ◽  
Effective Drugs

Ubiquitin-conjugating enzymes (E2s) are one of the three enzymes required by the ubiquitin-proteasome pathway to connect activated ubiquitin to target proteins via ubiquitin ligases. E2s determine the connection type of the ubiquitin chains, and different types of ubiquitin chains regulate the stability and activity of substrate proteins. Thus, E2s participate in the regulation of a variety of biological processes. In recent years, the importance of E2s in human health and diseases has been particularly emphasized. Studies have shown that E2s are dysregulated in variety of cancers, thus it might be a potential therapeutic target. However, the molecular basis of E2s as a therapeutic target has not been described systematically. We reviewed this issue from the perspective of the special position and role of E2s in the ubiquitin-proteasome pathway, the structure of E2s and biological processes they are involved in. In addition, the inhibitors and microRNAs targeting E2s are also summarized. This article not only provides a direction for the development of effective drugs but also lays a foundation for further study on this enzyme in the future.

scholarly journals SPZ1 promotes deregulation of Bim to boost apoptosis resistance in colorectal cancer

2020 ◽  
Vol 134 (2) ◽  
pp. 155-167
Author(s):  
Xiao-Yu Liu ◽  
Chang-Bo Zheng ◽  
Teng Wang ◽  
Jian Xu ◽  
Meng Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Growth ◽  
Clinical Stage ◽  
In Vivo Studies ◽  
Apoptosis Resistance ◽  
Advanced Clinical Stage ◽  
Helix Loop Helix ◽  
The Stability

Abstract Colorectal cancer (CRC) is the third most common malignancies in adults. Similar to other solid tumors, CRC cells show increased proliferation and suppressed apoptosis during the development and progression of the disease. Previous studies have shown that a novel tumor oncogene, spermatogenic basic helix-loop-helix transcription factor zip 1 (SPZ1), can promote proliferation. However, it is unclear whether SPZ1 plays a role in suppressing apoptosis, and the molecular mechanism behind SPZ1’s suppression of apoptosis in CRC remains unclear. Here, we found that silencing endogenous SPZ1 inhibits cell growth and induces apoptosis, and overexpression of SPZ1 promotes cell growth. These findings were corroborated by in vitro and in vivo studies. Interestingly, SPZ1 overexpressing cells were resistant to 5-fluorouracil, a drug commonly used to treat cancer. Moreover, knocking down SPZ1 led to the activation of caspase through the deregulation of Bim by ERK1/2, we found that CRC tissues had significantly higher SPZ1 and lower Bim expression, and SPZ1HBimL were associated with advanced clinical stage of CRC. Collectively, our findings demonstrate that SPZ1 contributes to tumor progression by limiting apoptosis. SPZ1 reduces apoptosis by altering the stability of Bim, suggesting SPZ1 may serve as a biomarker and therapeutic target for CRC.

scholarly journals Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide

1998 ◽  
Vol 333 (1) ◽  
pp. 99-105 ◽  
Author(s):  
Charles S. COCKELL ◽  
Julian M. MARSHALL ◽  
Keith M. DAWSON ◽  
Stewart A. CEDERHOLM-WILLIAMS ◽  
Chris P. PONTING
Keyword(s):  
Binding Site ◽  
Intramolecular Interaction ◽  
Current Model ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Physical Interaction ◽  
Kringle 5 ◽  
Type Plasminogen Activator ◽  
Lysine Residues

Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (α) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.

scholarly journals Poly(ADP-ribosyl)ating pathway regulates development from stem cell niche to longevity control

2021 ◽  
Vol 5 (3) ◽  
pp. e202101071
Author(s):  
Guillaume Bordet ◽  
Elena Kotova ◽  
Alexei V Tulin
Keyword(s):  
Posttranslational Modification ◽  
Stem Cell Niche ◽  
Egg Production ◽  
Phosphorylation Sites ◽  
Germline Stem Cells ◽  
Protein Activity ◽  
Cell Niche ◽  
Sufficient Food ◽  
Embryonic And Larval Development

The regulation of poly(ADP-ribose) polymerase, the enzyme responsible for the synthesis of homopolymer ADP-ribose chains on nuclear proteins, has been extensively studied over the last decades for its involvement in tumorigenesis processes. However, the regulation of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for removing this posttranslational modification, has attracted little attention. Here we identified that PARG activity is partly regulated by two phosphorylation sites, ph1 and ph2, in Drosophila. We showed that the disruption of these sites affects the germline stem-cells maintenance/differentiation balance as well as embryonic and larval development, but also the synchronization of egg production with the availability of a calorically sufficient food source. Moreover, these PARG phosphorylation sites play an essential role in the control of fly survivability from larvae to adults. We also showed that PARG is phosphorylated by casein kinase 2 and that this phosphorylation seems to protect PARG protein against degradation in vivo. Taken together, these results suggest that the regulation of PARG protein activity plays a crucial role in the control of several developmental processes.

scholarly journals Interaction between opposing modes of phospho-regulation of the proneural proteins Ascl1 and Ngn2

2018 ◽  
Vol 3 ◽  
pp. 129
Author(s):  
Laura J.A. Hardwick ◽  
Anna Philpott
Keyword(s):  
Inhibitory Effect ◽  
Protein Activity ◽  
Diverse Range ◽  
Temporal Precision ◽  
Temporal Regulation ◽  
Activating Mutations ◽  
Bhlh Domain ◽  
Helix Loop Helix ◽  
Progenitor Cell Proliferation

From the relatively simple nervous system of Drosophila to the elaborate mammalian cortex, neurogenesis requires exceptional spatial and temporal precision to co-ordinate progenitor cell proliferation and subsequent differentiation to a diverse range of neurons and glia. A limited number of transiently expressed proneural basic-helix-loop-helix (bHLH) transcription factors, for example achaete-scute-complex (as-c) and atonal (ato) in Drosophila and the vertebrate homologues Ascl1 and Neurogenin2 (Ngn2), are able to orchestrate the onset of neuronal determination, context-dependent subtype selection and even influence later aspects of neuronal migration and maturation. Within the last decade, two models have emerged to explain how the temporal activity of proneural determination factors is regulated by phosphorylation at distinct sites. One model describes how cell-cycle associated phosphorylation on multiple sites in the N and C termini of vertebrate proneural proteins limits neuronal differentiation in cycling progenitor cells. A second model describes phosphorylation on a single site in the bHLH domain of Drosophila atonal that acts as a binary switch, where phosphorylation terminates proneural activity. Here we combine activating mutations of phosphorylation sites in the N- and C- termini with an inhibitory phospho-mimetic mutation in the bHLH domain of Ascl1 and Ngn2 proteins, and test their functions in vivo using Xenopus embryos to determine which mode of phospho-regulation dominates. Enhancing activity by preventing N- and C terminal phosphorylation cannot overcome the inhibitory effect of mimicking phosphorylation of the bHLH domain. Thus we have established a hierarchy between these two modes of proneural protein control and suggest a model of temporal regulation for proneural protein activity.

scholarly journals Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

2020 ◽  
Author(s):  
Lukas Weiß ◽  
Tina Reiner ◽  
Julia Mergner ◽  
Bernhard Kuster ◽  
Attila Fehér ◽  
...  
Keyword(s):  
Amino Acid ◽  
Posttranslational Modification ◽  
Individual Case ◽  
Bound State ◽  
Amino Acid Residues ◽  
Nucleotide Exchange ◽  
Protein Activity ◽  
Developmental Defects

AbstractSmall RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major pathways regulate G-protein activity: canonical switching of the nucleotide bound state and posttranslational modification (PTM). PTMs can support or suppress RHO signaling, depending on each individual case. In plants, regulation of Rho of plants (ROPs) has been shown to act through nucleotide exchange and hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP Binding Kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is present in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa. Since this highly conserved amino acid residue is likewise ubiquitinated in mammalian Rac1 and Rac3, we suggest that RHO family proteins from different kingdoms could be generally regulated by ubiquitination of this site.

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