scholarly journals AMSH is an endosome-associated ubiquitin isopeptidase

2004 ◽  
Vol 166 (4) ◽  
pp. 487-492 ◽  
Author(s):  
John McCullough ◽  
Michael J. Clague ◽  
Sylvie Urbé
Keyword(s):  
Egf Receptor ◽  
Sh3 Domain ◽  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Isopeptidase Activity ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Inactivating Mutation ◽  
Free Ubiquitin

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif has been proposed to provide the active site for isopeptidase activity associated with the Rpn11/POH1 subunit of the 19S-proteasome and the Csn5-subunit of the signalosome. We have looked for similar activity in associated molecule with the SH3 domain of STAM (AMSH), a JAMM domain–containing protein that associates with the SH3-domain of STAM, a protein, which regulates receptor sorting at the endosome. We demonstrate isopeptidase activity against K48-linked tetraubiquitin and K63-linked polyubiquitin chains to generate di-ubiquitin and free ubiquitin, respectively. An inactivating mutation (D348A) in AMSH leads to accumulation of ubiquitin on endosomes and the concomitant stabilization of a ubiquitinated form of STAM, which requires an intact ubiquitin interaction motif (UIM) within STAM. Short interfering RNA knockdown of AMSH enhances the degradation rate of EGF receptor (EGFR) following acute stimulation and ubiquitinated EGFR provides a substrate for AMSH in vitro. We propose that AMSH is a deubiquitinating enzyme with functions at the endosome, which oppose the ubiquitin-dependent sorting of receptors to lysosomes.

scholarly journals Targeted Deletion of the Murine apobec-1 Complementation Factor (acf) Gene Results in Embryonic Lethality

2005 ◽  
Vol 25 (16) ◽  
pp. 7260-7269 ◽  
Author(s):  
Valerie Blanc ◽  
Jeffrey O. Henderson ◽  
Elizabeth P. Newberry ◽  
Susan Kennedy ◽  
Jianyang Luo ◽  
...  
Keyword(s):  
Rna Binding ◽  
Cytidine Deaminase ◽  
Mrna Editing ◽  
Targeted Deletion ◽  
Apob Mrna ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Binding Subunit ◽  
Apparently Healthy

ABSTRACT apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf +/ − mice were apparently healthy and fertile, no viable acf − / − mice were identified. Mutant acf − / − embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf − / − blastocysts were detectable following implantation at E4.5, and isolated acf − / − blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

scholarly journals Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 896 ◽  
Author(s):  
Shaochuan Zhou ◽  
Xinna Ge ◽  
Can Kong ◽  
Teng Liu ◽  
Aijing Liu ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Biochemical Properties ◽  
In Vitro Assays ◽  
Respiratory Syndrome Virus ◽  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Cleavage Activity ◽  
Tnf Α ◽  
Cis And Trans

The papain-like cysteine protease 2 (PLP2) within the N-terminus of the porcine reproductive and respiratory syndrome virus (PRRSV) nsp2 replicase protein specifies a deubiquitinating enzyme (DUB), but its biochemical properties and the role in infection have remained poorly defined. By using in vitro assays, we found that the purified PLP2 could efficiently cleave K63 and K48 linked polyubiquitin chains Ub3-7 in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. The subsequent mutagenesis analyses revealed that the requirement for PLP2 DUB activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., D91R, D85R, etc.) that largely ablated the DUB function also blocked the cis- but not trans-proteolytic cleavage of nsp2/3 polyprotein. Moreover, the analyses identified key mutations that could differentiate DUB from PLP2 cis- and trans-cleavage activities. Further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the DUB activity were all lethal to the virus, (ii) a point mutation T88G that selectively blocked the cis-cleavage activity of PLP2 did not affect viral viability in cell culture, and (iii) an E90Q mutation that did not affect either of the PLP2 activities led to rescue of WT-like virus but displayed significantly reduced ability to induce TNF-α production. Our findings support the possibility that the PLP2 DUB activity, but not cis-cleavage activity, is essential for PRRSV replication. The data also establish a strong link of nsp2 to pro-inflammatory cytokine induction during infection that operates in a manner independent of PLP2 DUB activity.

scholarly journals Characterization of OTUB1 activation and inhibition by different E2 enzymes

2019 ◽  
Author(s):  
Lauren T. Que ◽  
Marie E. Morrow ◽  
Cynthia Wolberger
Keyword(s):  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Kinetics And Thermodynamics ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Stimulation Of

AbstractOTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.

scholarly journals Amino Acid Residues Required for Physical and Cooperative Transcriptional Interaction of STAT3 and AP-1 Proteins c-Jun and c-Fos

2007 ◽  
Vol 27 (18) ◽  
pp. 6300-6308 ◽  
Author(s):  
Michael Ginsberg ◽  
Elmar Czeko ◽  
Patrick Müller ◽  
Zhiyong Ren ◽  
Xiaomin Chen ◽  
...  
Keyword(s):  
Target Genes ◽  
Amino Acid Residues ◽  
Chromosomal Dna ◽  
Protein Interfaces ◽  
Specific Contact ◽  
Dna Elements ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Chromosomal Loci

ABSTRACT Cooperation between STAT3 and c-Jun in driving transcription during transfection of reporter constructs is well established, and both proteins are present on some interleukin-6 (IL-6) STAT3-dependent promoters on chromosomal loci. We report that small interfering RNA knockdown of c-Jun or c-Fos diminishes IL-6 induction of some but not all STAT3-dependent mRNAs. Specific contact sites in STAT3 responsible for interaction of a domain of STAT3 with c-Jun were known. Here we show that the B-zip domain of c-Jun interacts with STAT3 and that c-Jun mutation R261A or R261D near but not in the DNA binding domain blocks in vitro STAT3-c-Jun interaction and decreases costimulation of transcription in transfection assays. Cooperative binding to DNA of tyrosine-phosphorylated STAT3 and both wild-type and R261A mutant c-Jun was observed. Even c-Jun mutant R261D, which on its own did not bind DNA, bound DNA weakly in the presence of STAT3. We conclude that a functional interaction between STAT3 and c-Jun while bound to chromosomal DNA elements exists and is necessary for driving transcription on at least some STAT3 target genes. Identifying such required interactive protein interfaces should be a stimulus to search for compounds that could ultimately inhibit the activity of STAT3 in tumors dependent on persistently active STAT3.

scholarly journals Endoplasmic Reticulum (ER) Mannosidase I Is Compartmentalized and Required for N-Glycan Trimming to Man5–6GlcNAc2 in Glycoprotein ER-associated Degradation

2008 ◽  
Vol 19 (1) ◽  
pp. 216-225 ◽  
Author(s):  
Edward Avezov ◽  
Zehavit Frenkel ◽  
Marcelo Ehrlich ◽  
Annette Herscovics ◽  
Gerardo Z. Lederkremer
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Local Concentration ◽  
Mannose Residue ◽  
Low Concentrations ◽  
High Concentrations ◽  
Rna Knockdown ◽  
Interfering Rna

We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man9GlcNAc2. A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to four α1,2-linked mannose residues. Using small interfering RNA knockdown of ERManI, we show that this enzyme is required for trimming to Man5–6GlcNAc2 and for ERAD in cells in vivo, leading to the accumulation of Man9GlcNAc2 and Glc1Man9GlcNAc2 on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI is strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knockdown prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation.

SiRNA technology, the gene therapy of the future?

2008 ◽  
Vol 149 (4) ◽  
pp. 153-159 ◽  
Author(s):  
Zsuzsanna Rácz ◽  
Péter Hamar
Keyword(s):  
Gene Therapy ◽  
Short Interfering Rna ◽  
The Future ◽  
Interfering Rna

A genetikában új korszak kezdődött 17 éve, amikor a petúniában felfedezték a koszuppressziót. Később a koszuppressziót azonosították a növényekben és alacsonyabb rendű eukariótákban megfigyelt RNS-interferenciával (RNSi). Bár a növényekben ez ősi vírusellenes gazdaszervezeti védekezőmechanizmus, emlősökben az RNSi élettani szerepe még nincs teljesen tisztázva. Az RNSi-t rövid kettős szálú interferáló RNS-ek (short interfering RNA, siRNS) irányítják. A jelen cikkben összefoglaljuk az RNSi történetét és mechanizmusát, az siRNS-ek szerkezete és hatékonysága közötti összefüggéseket, a célsejtbe való bejuttatás virális és nem virális módjait. Az siRNS-ek klinikai alkalmazásának legfontosabb akadálya az in vivo alkalmazás. Bár a hidrodinamikus kezelés állatokban hatékony, embereknél nem alkalmazható. Lehetőséget jelent viszont a szervspecifikus katéterezés. A szintetizált siRNS-ek ismert mellékhatásait szintén tárgyaljuk. Bár a génterápia ezen új területén számos problémával kell szembenézni, a sikeres in vitro és in vivo kísérletek reményt jelentenek emberi betegségek siRNS-sel történő kezelésére.

In vitro binding activity between HCK SH3 domain and HIV-1 Nef protein

2011 ◽  
Vol 31 (3) ◽  
pp. 262-265
Author(s):  
Xiao-lin QIN ◽  
Chao-qi LIU ◽  
Dong-ming REN ◽  
Yong-qin ZHOU
Keyword(s):  
Sh3 Domain ◽  
Binding Activity ◽  
Hiv 1

Met -/- kidneys express epithelial cells that chemotax and form tubules in response to EGF receptor ligands

1997 ◽  
Vol 272 (2) ◽  
pp. F222-F228
Author(s):  
C. Kjelsberg ◽  
H. Sakurai ◽  
K. Spokes ◽  
C. Birchmeier ◽  
I. Drummond ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cell ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Receptor Ligands ◽  
Tubule Formation ◽  
Human Papillomavirus 16 ◽  
Immortalized Cells

The growth factor/receptor combination of hepatocyte growth factor (HGF)/c-met has been postulated to be critical for mesenchymal-to-epithelial conversion and tubule formation in the developing kidney. We therefore isolated and immortalized cells from embryonic kidneys of met -/- transgenic mice to determine whether these cells were epithelial and able to chemotax and form tubules in vitro. The cells were immortalized with retrovirus expressing human papillomavirus 16 (HPV 16) E6/E7 genes. Two rapidly dividing clones were isolated and found to express the epithelial cell markers cytokeratin, zonula occludens-1, and E-cadherin but not to express the fibroblast marker vimentin. The met -/- cells were able to chemotax in response to epidermal growth factor and transforming growth factor-alpha (TGF-alpha) and form tubules in vitro in response to TGF-alpha but not HGF. These experiments suggest that the HGF/c-met axis is not essential for epithelial cell development in the embryonic kidney and demonstrate that other growth factors are capable of supporting early tubulogenesis.

scholarly journals GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment

2019 ◽  
Vol 4 (42) ◽  
pp. eaax8189 ◽  
Author(s):  
Marie Jo Halaby ◽  
Kebria Hezaveh ◽  
Sara Lamorte ◽  
M. Teresa Ciudad ◽  
Andreas Kloetgen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Macrophage Polarization ◽  
Suppressor Cells ◽  
Interferon Γ ◽  
Mass Cytometry ◽  
General Control ◽  
Immune Attack ◽  
Environmental Sensor ◽  
Rna Knockdown ◽  
Interfering Rna

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor–binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

scholarly journals Autophagy induction in atrophic muscle cells requires ULK1 activation by TRIM32 through unanchored K63-linked polyubiquitin chains

2019 ◽  
Vol 5 (5) ◽  
pp. eaau8857 ◽  
Author(s):  
M. Di Rienzo ◽  
M. Antonioli ◽  
C. Fusco ◽  
Y. Liu ◽  
M. Mari ◽  
...  
Keyword(s):  
Mouse Models ◽  
Muscle Atrophy ◽  
Ubiquitin Ligase ◽  
Muscle Dystrophy ◽  
Polyubiquitin Chains ◽  
Autophagy Induction ◽  
Atrophic Muscle ◽  
Autophagic Activity

Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32’s ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.

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