scholarly journals The Ulp1 SUMO isopeptidase

2003 ◽  
Vol 160 (7) ◽  
pp. 1069-1082 ◽  
Author(s):  
Shyr-Jiann Li ◽  
Mark Hochstrasser
Keyword(s):  
Protein Modification ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Protease Domain ◽  
Cycle Progression ◽  
Terminal Domain ◽  
Specific Protease ◽  
Cysteine Protease Domain ◽  
Necessary And Sufficient

Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A ∼200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

scholarly journals Endothelial cells isolated from caveolin-2 knockout mice display higher proliferation rate and cell cycle progression relative to their wild-type counterparts

2010 ◽  
Vol 298 (3) ◽  
pp. C693-C701 ◽  
Author(s):  
Leike Xie ◽  
Philippe G. Frank ◽  
Michael P. Lisanti ◽  
Grzegorz Sowa
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Inhibitors ◽  
Fold Increase ◽  
S Phase ◽  
Lower Percentage ◽  
Wild Type ◽  
Cycle Progression ◽  
Molecular Events

The goal of this study was to determine whether caveolin-2 (Cav-2) is capable of controlling endothelial cell (EC) proliferation in vitro. To realize this goal, we have directly compared proliferation rates and cell cycle-associated signaling proteins between lung ECs isolated from wild-type (WT) and Cav-2 knockout (KO) mice. Using three independent proliferation assays, we have determined that Cav-2 KO ECs proliferate by ca. 2-fold faster than their WT counterparts. Cell cycle analysis by flow cytometry of propidium iodide-stained cells showed a relatively higher percentage of Cav-2 KO ECs in S and G2/M and lower percentage in Go/G1 phases of cell cycle relative to their WT counterparts. Furthermore, an over 2-fold increase in the percentage of S phase-associated Cav-2 KO relative to WT ECs was independently determined with bromodeoxyuridine incorporation assay. Mechanistically, the increase in proliferation/cell cycle progression of Cav-2 KO ECs correlated well with elevated expression levels of predominantly S phase- and G2/M phase-associated cyclin A and B1, respectively. Further mechanistic analysis of molecular events controlling cell cycle progression revealed increased level of hyperphosphorylated (inactive) form of G1 to S phase transition inhibitor, the retinoblastoma protein in hyperproliferating Cav-2 KO ECs. Conversely, the expression level of the two cyclin-dependent kinase inhibitors p16INK4 and p27Kip1 was reduced in Cav-2 KO ECs. Finally, increased phosphorylation (activation) of proproliferative extracellular signal-regulated kinase 1/2 was observed in hyperproliferating Cav-2 KO ECs. Overall, our data suggest that Cav-2 negatively regulates lung EC proliferation and cell cycle progression.

scholarly journals Crystal structure of the SENP1 mutant C603S–SUMO complex reveals the hydrolytic mechanism of SUMO-specific protease

2006 ◽  
Vol 398 (3) ◽  
pp. 345-352 ◽  
Author(s):  
Zheng Xu ◽  
So Fun Chau ◽  
Kwok Ho Lam ◽  
Ho Yin Chan ◽  
Tzi Bun Ng ◽  
...  
Keyword(s):  
Active Site ◽  
Cell Cycle Progression ◽  
Catalytic Domain ◽  
Cysteine Residue ◽  
Cycle Progression ◽  
Vitro Assay ◽  
Active Site Cysteine ◽  
Specific Protease ◽  
Hydrolytic Mechanism

SUMO (small ubiquitin-related modifier)-specific proteases catalyse the maturation and de-conjugation processes of the sumoylation pathway and modulate various cellular responses including nuclear metabolism and cell cycle progression. The active-site cysteine residue is conserved among all known SUMO-specific proteases and is not substitutable by serine in the hydrolysis reactions demonstrated previously in yeast. We report here that the catalytic domain of human protease SENP1 (SUMO-specific protease 1) mutant SENP1CC603S carrying a mutation of cysteine to serine at the active site is inactive in maturation and de-conjugation reactions. To further understand the hydrolytic mechanism catalysed by SENP1, we have determined, at 2.8 Å resolution (1 Å=0.1 nm), the X-ray structure of SENP1CC603S–SUMO-1 complex. A comparison of the structure of SENP2–SUMO-1 suggests strongly that SUMO-specific proteases require a self-conformational change prior to cleavage of peptide or isopeptide bond in the maturation and de-conjugation processes respectively. Moreover, analysis of the interface of SENP1 and SUMO-1 has led to the identification of four unique amino acids in SENP1 that facilitate the binding of SUMO-1. By means of an in vitro assay, we further demonstrate a novel function of SENP1 in hydrolysing the thioester linkage in E1-SUMO and E2-SUMO complexes. The results disclose a new mechanism of regulation of the sumoylation pathway by the SUMO-specific proteases.

scholarly journals Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

2008 ◽  
Vol 19 (12) ◽  
pp. 5203-5213 ◽  
Author(s):  
Isabella Manni ◽  
Giuseppina Caretti ◽  
Simona Artuso ◽  
Aymone Gurtner ◽  
Velia Emiliozzi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Transcriptional Activity ◽  
Regulatory Subunit ◽  
Posttranslational Regulation ◽  
Wild Type ◽  
Cycle Progression ◽  
Mitotic Cyclin ◽  
Wild Type Form

NF-Y binds to CCAAT motifs in the promoter region of a variety of genes involved in cell cycle progression. The NF-Y complex comprises three subunits, NF-YA, -YB, and -YC, all required for DNA binding. Expression of NF-YA fluctuates during the cell cycle and is down-regulated in postmitotic cells, indicating its role as the regulatory subunit of the complex. Control of NF-YA accumulation is posttranscriptional, NF-YA mRNA being relatively constant. Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity.

p38α Activates Purine Metabolism to Initiate Hematopoietic Stem Cell Cycling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 776-776
Author(s):  
Daiki Karigane ◽  
Shinichiro Okamoto ◽  
Toshio Suda ◽  
Keiyo Takubo
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Purine Metabolism ◽  
Stress Conditions ◽  
Wild Type ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. However, how stress hematopoiesis, including bone marrow transplantation (BMT), induces metabolic changes in HSCs remains unclear. Here, we report a critical role for the p38MAPK family isoform p38α in initiating HSC proliferation during stress hematopoiesis in mouse. First, we identified p38α as the major p38MAPK isozyme highly expressed in HSCs and we also performed conditional knockout of p38α in mice. This mouse showed no overt difference relative to wild type mouse. However, treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type mice (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type mice, N=6, p=0.03) and loss of p38α in HSCs showed a defective transplantation capacity in primary and secondary transplantation. To gain further insight into p38MAPK function during hematological stress, we evaluated the time course of p38MAPK activation in stressful contexts by intracellular flow cytometry. We found that p38MAPK was immediately phosphorylated in HSCs after hematological stress and returned to normal in a short period, suggesting that p38α functions rapidly after hematological stresses to activate downstream events. To identify events downstream of p38α after hematological stress, we initially evaluated mechanisms such as homing, apoptosis, and ROS generation immediately after BMT. However, defects seen in p38α-deficient HSCs after hematological stress could not be explained by these mechanisms. Therefore we next focused on cell cycle. In CFSE assay, p38α loss resulted in defective recovery from hematological stress and a delay in initiating cycling of HSCs. In addition, p38α-deficient HSCs showed lower BrdU incorporation in vivo (p=0.045) and EdU incorporation in vitro (p=0.003). Transcriptome analysis of transplanted wild-type or p38α-deficient HSCs suggested that p38α-deficient HSCs showed lower enrichment of genes related to HSC-related markers and proliferation. Taken together, loss of p38α resulted in defective HSC cell cycle progression in stressed settings such as transplantation. Given that altered metabolic activities can change cell cycle status, we asked whether p38α regulation of a particular metabolic pathway could initiate HSC cycling under stress conditions. To do so, we collected p38α-deficient or wild-type LSK cells either at steady state or after BMT and extracted metabolites for metabolome analysis using mass spectrometry. Among metabolites surveyed, we focused on changes in glycine and aspartic acid, which are required for purine biosynthesis. Levels of both increased in p38α-deficient as compared with wild-type LSK cells after BMT. Also, mice transplanted with p38α-deficient compared with wild-type LSK cells showed lower levels of allantoin, a product of purine catabolism. These findings suggest that p38α loss suppresses purine metabolism during stress hematopoiesis. Next, we evaluated mRNAs encoding key enzymes functioning in purine metabolism by qPCR. Expression of both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), and guanosine monophosphate synthetase (GMPS) was significantly decreased in p38α-deficient HSCs relative to wild-type HSCs on day 1 after BMT. To assess how changes in purine metabolism could affect the HSC response to stress, we treated HSCs with cytokines in the presence or absence of mycophenolic acid (MPA), an IMPDH2 inhibitor. MPA treatment significantly suppressed colony formation capacity of HSCs in a dose-dependent manner. Also, EdU incorporation into HSCs was reduced by MPA dose-dependently. Finally, isolated HSCs were cultured with or without MPA for 3 days and then transplanted into recipients along with competitor cells. PB chimerism was dose-dependently decreased in recipients of MPA-treated cells. These findings suggest that purine metabolism directly maintains proliferation capacity of HSCs in stress conditions. In summary, expression of purine-synthesizing enzymes decreased in p38α-deficient HSCs after transplantation, an activity correlated with defective cell cycle progression in vitro and in vivo. Overall, this is the first report of p38α-regulated changes in purine metabolism associated with HSC stress and cell cycle initiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tryprostatin A, a specific and novel inhibitor of microtubule assembly

1998 ◽  
Vol 333 (3) ◽  
pp. 543-548 ◽  
Author(s):  
Takeo USUI ◽  
Masuo KONDOH ◽  
Cheng-Bin CUI ◽  
Tadanori MAYUMI ◽  
Hiroyuki OSADA
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Progression ◽  
Microtubule Assembly ◽  
Inhibition Mechanism ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Terminal Domain ◽  
M Phase

We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 µM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 µM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.

Identification of the human Nα-acetyltransferase complex B (hNatB): a complex important for cell-cycle progression

2008 ◽  
Vol 415 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Kristian K. Starheim ◽  
Thomas Arnesen ◽  
Darina Gromyko ◽  
Anita Ryningen ◽  
Jan Erik Varhaug ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Normal Cell ◽  
Protein Modification ◽  
Cell Cycle Progression ◽  
Cell Function ◽  
Cancer Cell Line ◽  
G1 Phase ◽  
Stable Complex ◽  
Cycle Progression

Protein Nα-terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, protein Nα-terminal acetylation is performed by the N-acetyltransferase complexes NatA, NatB and NatC. In humans, only the NatA complex has been identified and characterized. In the present study we present the components of hNatB (human NatB complex). It consists of the Nat3p homologue hNAT3 (human N-acetyltransferase 3) and the Mdm20p homologue hMDM20 (human mitochondrial distribution and morphology 20). They form a stable complex and in vitro display sequence-specific Nα-acetyltransferase activity on a peptide with the N-terminus Met-Asp-. hNAT3 and hMDM20 co-sediment with ribosomal pellets, thus supporting a model where hNatB acts co-translationally on nascent polypeptides. Specific knockdown of hNAT3 and hMDM20 disrupts normal cell-cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL-62. hNAT3 knockdown results in an increase in G0/G1-phase cells, whereas hMDM20 knockdown decreased the fraction of cells in G0/G1-phase and increased the fraction of cells in the sub-G0/G1-phase. In summary, we show for the first time a vertebrate NatB protein Nα-acetyltransferase complex essential for normal cell proliferation.

scholarly journals Structural basis of Stu2 recruitment to yeast kinetochores

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jacob A Zahm ◽  
Michael G Stewart ◽  
Joseph S Carrier ◽  
Stephen C Harrison ◽  
Matthew P Miller
Keyword(s):  
Cell Cycle Progression ◽  
Terminal Segment ◽  
Structural Basis ◽  
Wild Type ◽  
Cycle Progression ◽  
Delay Cell ◽  
Sister Chromatids

Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these ‘bioriented’ sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.

scholarly journals RACK1 Specifically Regulates Translation through Its Binding to Ribosomes

2018 ◽  
Vol 38 (23) ◽  
Author(s):  
Simone Gallo ◽  
Sara Ricciardi ◽  
Nicola Manfrini ◽  
Elisa Pesce ◽  
Stefania Oliveto ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Cell Cycle Progression ◽  
Living Cells ◽  
Initiation Factor ◽  
Wild Type ◽  
Physiological Mechanisms ◽  
Cycle Progression ◽  
Eukaryotic Initiation Factor 4E ◽  
Kinase C

ABSTRACTThe translational capability of ribosomes deprived of specific nonfundamental ribosomal proteins may be altered. Physiological mechanisms are scanty, and it is unclear whether free ribosomal proteins can cross talk with the signaling machinery. RACK1 (receptor for activated C kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling, such as the protein kinase C (PKC) family. Here we show that the binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eukaryotic initiation factor 4E (eIF4E).In vitro, when RACK1 is partially depleted, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas the addition of a RACK1 mutant that is unable to bind ribosomes does not. Outside the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype, impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly through interaction with known partners on or off the ribosome that are involved in signaling.

scholarly journals LMNB2 promotes the progression of colorectal cancer by silencing p21 expression

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Chen-Hua Dong ◽  
Tao Jiang ◽  
Hang Yin ◽  
Hu Song ◽  
Yi Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Gene Set Enrichment Analysis ◽  
Molecular Therapy ◽  
Cycle Progression ◽  
Cancer Tissues

AbstractColorectal cancer is the second common cause of death worldwide. Lamin B2 (LMNB2) is involved in chromatin remodeling and the rupture and reorganization of nuclear membrane during mitosis, which is necessary for eukaryotic cell proliferation. However, the role of LMNB2 in colorectal cancer (CRC) is poorly understood. This study explored the biological functions of LMNB2 in the progression of colorectal cancer and explored the possible molecular mechanisms. We found that LMNB2 was significantly upregulated in primary colorectal cancer tissues and cell lines, compared with paired non-cancerous tissues and normal colorectal epithelium. The high expression of LMNB2 in colorectal cancer tissues is significantly related to the clinicopathological characteristics of the patients and the shorter overall and disease-free cumulative survival. Functional analysis, including CCK8 cell proliferation test, EdU proliferation test, colony formation analysis, nude mouse xenograft, cell cycle, and apoptosis analysis showed that LMNB2 significantly promotes cell proliferation by promoting cell cycle progression in vivo and in vitro. In addition, gene set enrichment analysis, luciferase report analysis, and CHIP analysis showed that LMNB2 promotes cell proliferation by regulating the p21 promoter, whereas LMNB2 has no effect on cell apoptosis. In summary, these findings not only indicate that LMNB2 promotes the proliferation of colorectal cancer by regulating p21-mediated cell cycle progression, but also suggest the potential value of LMNB2 as a clinical prognostic marker and molecular therapy target.

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