scholarly journals An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint

2019 ◽  
Author(s):  
Ben F. Brian ◽  
Myra G. Nunez ◽  
Kathryn L. Schwertfeger ◽  
Tanya S. Freedman
Keyword(s):  
Steady State ◽  
Ubiquitin Ligase ◽  
Biochemical Analysis ◽  
Cell Activation ◽  
Critical Factor ◽  
Myeloid Cell ◽  
State Level ◽  
Cell Specificity ◽  
Steady State Levels ◽  
Insert Region

AbstractThe activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is critical factor regulating myeloid-cell activation. In a previous paper (Freedman et al., 2015) we showed in macrophages that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation, functioning as a rheostat regulating ITAM signaling. We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic and biochemical analysis, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via tyrosine 32 in its unique insert region. This orthogonal mode of c-Cbl recognition depresses the steady-state level of macrophage LynA. Mast cells, however, express little c-Cbl and have correspondingly high steady-state levels of LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression therefore builds cell specificity into the LynA checkpoint.

scholarly journals Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ben F Brian ◽  
Adrienne S Jolicoeur ◽  
Candace R Guerrero ◽  
Myra G Nunez ◽  
Zoi E Sychev ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Cell Activation ◽  
E3 Ubiquitin Ligase ◽  
Critical Factor ◽  
Myeloid Cell ◽  
Src Family Kinases ◽  
Distinct Mode ◽  
Unique Region ◽  
Rapid Degradation ◽  
Cell Specificity

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles

2006 ◽  
Vol 80 (12) ◽  
pp. 5886-5896 ◽  
Author(s):  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Steady State ◽  
Human Cytomegalovirus ◽  
Virus Titer ◽  
State Level ◽  
Early Gene ◽  
Tegument Protein ◽  
Cyclin Dependent Kinase ◽  
Viral Dna ◽  
Infected Cells ◽  
Steady State Levels

ABSTRACT We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

IL-3 and Retinoic Acid-Induced Myeloid Differentiation of Hematopoietic Progenitors Is Negatively Regulated by E3 Ubiquitin Ligase Rnf41 (FLRF).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3160-3160
Author(s):  
Xin Jing ◽  
Luis Dabul ◽  
Ronald G. Nachtman ◽  
Roland Jurecic
Keyword(s):  
Retinoic Acid ◽  
Steady State ◽  
Ligand Binding ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Myeloid Differentiation ◽  
Hematopoietic Progenitors ◽  
Induced Differentiation ◽  
Gm Csf ◽  
Steady State Levels

Abstract The proliferation and differentiation of hematopoietic cells is regulated by interaction of diverse group of cytokines with corresponding receptors. Regulation of cytokine signaling occurs at multiple levels, including the regulation of the amount of receptors present before and after ligand binding. However, the mechanisms that govern pre-determined steady-state amount of cytokine receptors are poorly understood. Previously we have reported that new E3 ubiquitin ligase Rnf41 (FLRF/Nrdp1) regulates cytokine-induced differentiation of hematopoietic progenitors through negative regulation of steady-state receptor levels. Increased levels of Rnf41 protein significantly attenuate differentiation of multipotent progenitors in response to IL-3, Epo and GM-CSF, due to a significant and constitutive decrease in the steady-state amount of IL-3, Epo and GM-CSF receptors. Immunoprecipitation and Western analysis of proteins from several types of hematopoietic progenitors has confirmed that Rnf41 protein associates with IL-3, Epo and GM-CSF receptors, and has shown that Rnf41-mediated down-regulation of receptors is independent of the ligand binding. Thus, by regulating steady-state amounts of IL-3, Epo and GM-CSF receptors Rnf41 could be maintaining optimal levels of signaling necessary for proper lineage commitment and differentiation of HSC and progenitors. Retinoic acid (RA) is known to augment IL-3 and GM-CSF induced differentiation of progenitors into myeloid lineages. Surprisingly, instead of improving the RA treatment further suppressed IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors over-expressing Rnf41. The RA functions through RARα and RXR receptors, whihc act as transcription factors and interact with specific DNA targets as hetero- (RAR-RXR) or homodimers (RXR-RXR). Interestingly, Western analysis has shown that hematopoietic progenitors over-expressing Rnf41 exhibit a reduction in the steady-state levels of RARα receptors, whereas levels of RXR receptors remain unchanged. Moreover, the treatment of hematopoietic progenitors over-expressing Rnf41 with RA leads to even further decrease of RARα receptor levels. The transient over-expression of Rnf41 in BaF3 cell line also decreases steady-state levels of RARα receptors, while RXR receptor levels remain unchanged. Protein IP with α-Rnf41, α-RARα or α-RXR antibodies has shown that endogenous Rnf41 protein associates with RARα receptors in hematopoietic progenitors. Taken together, these results suggest that Rnf41 influences RA-mediated myeloid differentiation of hematopoietic progenitors by negatively regulating the levels of RARα receptors. Several studies have reported that IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors leads to activation of RARa through induction of the Jak2/Stat5 pathway. These studies have defined a previously unknown cytokine-RAR interaction during myelopoiesis and suggested that RAR activation might be a critical downstream event following IL-3 and GM-CSF signaling during myeloid differentiation. Combined together the existing data suggest a model in which Rnf41 negatively regulates myeloid differentiation of hematopoietic progenitors by independently regulating steady state levels of IL-3, GM-CSF and RAR receptors, and thus impacting cytokine and RA signaling. Ongoing studies with RA agonists and antagonists are aimed at unraveling further the role of Rnf41 in regulation of RA signaling during progenitor cell differentiation.

scholarly journals Activation of virus replication after vaccination of HIV-1-infected individuals.

1995 ◽  
Vol 182 (6) ◽  
pp. 1727-1737 ◽  
Author(s):  
S I Staprans ◽  
B L Hamilton ◽  
S E Follansbee ◽  
T Elbeik ◽  
P Barbosa ◽  
...  
Keyword(s):  
T Cells ◽  
Steady State ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Activation ◽  
Cell Activation ◽  
Vaccine Antigens ◽  
Steady State Levels ◽  
Hiv 1

Little is known about the factors that govern the level of HIV-1 replication in infected individuals. Recent studies (using potent antiviral drugs) of the kinetics of HIV-1 replication in vivo have demonstrated that steady-state levels of viremia are sustained by continuous rounds of de novo infection and the associated rapid turnover of CD4+ T lymphocytes. However, no information is available concerning the biologic variables that determine the size of the pool of T cells that are susceptible to virus infection or the amount of virus produced from infected cells. Furthermore, it is not known whether all CD4+ T lymphocytes are equally susceptible to HIV-1 infection at a given time or whether the infection is focused on cells of a particular state of activation or antigenic specificity. Although HIV-1 replication in culture is known to be greatly facilitated by T cell activation, the ability of specific antigenic stimulation to augment HIV-1 replication in vivo has not been studied. We sought to determine whether vaccination of HIV-1-infected adults leads to activation of virus replication and the targeting of vaccine antigen-responsive T cells for virus infection and destruction. Should T cell activation resulting from exposure to environmental antigens prove to be an important determinant of the steady-state levels of HIV-1 replication in vivo and lead to the preferential loss of specific populations of CD4+ T lymphocytes, it would have significant implications for our understanding of and therapeutic strategies for HIV-1 disease. To begin to address these issues, HIV-1-infected individuals and uninfected controls were studied by measurement of immune responses to influenza antigens and quantitation of virion-associated plasma HIV-1 RNA levels at baseline and at intervals after immunization with the trivalent influenza vaccine. Influenza vaccination resulted in readily demonstrable but transient increases in plasma HIV-1 RNA levels, indicative of activation of viral replication, in HIV-1-infected individuals with preserved ability to immunologically respond to vaccine antigens. Activation of HIV-1 replication by vaccination was more often seen and of greater magnitude in individuals who displayed a T cell proliferative response to vaccine antigens at baseline and in those who mounted a significant serologic response after vaccination. The fold increase in viremia, as well as the rates of increase of HIV-1 in plasma after vaccination and rates of viral decline after peak viremia, were higher in individuals with higher CD4+ T cell counts.(ABSTRACT TRUNCATED AT 400 WORDS)

Steady-State Serum Concentrations of Dothiepin and Northiaden after Two Dosage Regimens of Dothiepin Hydrochloride (Prothiaden)

1973 ◽  
Vol 1 (2) ◽  
pp. 391-397
Author(s):  
B R S Nakra ◽  
R C Glass ◽  
J A Rees
Keyword(s):  
Steady State ◽  
Parent Drug ◽  
State Level ◽  
Serum Levels ◽  
Single Daily Dose ◽  
Serum Concentrations ◽  
Dosage Regimens ◽  
Daily Dose ◽  
Steady State Serum ◽  
Steady State Levels

Five healthy volunteers took part in a crossover study which examined the serum concentrations of dothiepin and northiaden after a 25 mg three times a day and a 75 mg once a day dosage regimen of Prothiaden. The inter-individual variation of serum levels was large after either schedule which is to be expected with this group of drugs. The minimum steady-state level of dothiepin tended to be lower after the single daily dose, but the differences were small and not statistically significant. The approximate maximum steady-state levels of dothiepin showed large intra- and inter-subject variation and no obvious trend. The values of the desmethylated metabolite, northiaden, tended to follow the dothiepin concentrations but were lower than the parent drug. Average steady-state levels tended, with one exception, to be very similar after both regimens with no evidence of any trend when comparing the two regimens. The study showed that the two regimens yielded similar steady-state serum concentrations both of drug and metabolite but inter-individual differences were large.

scholarly journals Structure-Function Study of MalF Protein by Random Mutagenesis

1999 ◽  
Vol 181 (7) ◽  
pp. 2267-2272 ◽  
Author(s):  
María Isabel Tapia ◽  
Michaël Mourez ◽  
Maurice Hofnung ◽  
Elie Dassa
Keyword(s):  
Steady State ◽  
Subcellular Localization ◽  
Cytoplasmic Membrane ◽  
Random Mutagenesis ◽  
State Level ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
Functional Regions ◽  
Large N ◽  
Steady State Levels

ABSTRACT MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malFmutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism.

scholarly journals Role of STE genes in the mating factor signaling pathway mediated by GPA1 in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (9) ◽  
pp. 3777-3783 ◽  
Author(s):  
N Nakayama ◽  
Y Kaziro ◽  
K Arai ◽  
K Matsumoto
Keyword(s):  
Steady State ◽  
Signaling Pathway ◽  
State Level ◽  
G1 Arrest ◽  
Gene Products ◽  
Mating Efficiency ◽  
Protein Functions ◽  
Steady State Levels ◽  
Mating Factor

The ste mutants (ste2, ste4, ste5, ste7, ste11, and ste12) are insensitive to mating factors and are, therefore, sterile. Roles of the STE gene products in the GPA1-mediated mating factor signaling pathway were studied by using ste gpa1 double mutants. Mating efficiency of a ste2 mutant defective in the alpha-factor receptor increased 1,000-fold in a gpa1 background, while G1 arrest and aberrant morphology (shmoo) caused by gpa1 were not suppressed by ste2. Furthermore, the steady-state level of the FUS1 transcript, which normally increases in response to mating factors, was also elevated when the GPA1 function was impaired. These results suggest that the GPA1 protein functions downstream of the STE2 receptor. Conversely, the sterility of ste4, ste5, ste7, ste11, and ste12 mutants was not suppressed by gpa1, but the lethal phenotype of gpa1 was suppressed by these ste mutations. Northern (RNA) blotting analysis revealed that the ste7, ste11, and ste12 mutations caused reductions of 50 to 70% in the steady-state levels of the GPA1 transcript, while ste4 had a slight effect and ste5 had no effect. This implies that the suppression by ste7, ste11, and ste12 could be due to reduced syntheses of additional components, including an effector, and that suppression by ste4 and ste5 may result from direct effects on the signaling pathway. The STE4, STE5, STE7, STE11, and STE12 products, therefore, appear to specify components of the signal transduction machinery, directly or indirectly, which function together with or downstream of GPA1.

scholarly journals NEMO score in nailfold videocapillaroscopy is a good tool to assess both steady state levels and overtime changes of disease activity in patients with systemic sclerosis: a comparison with the proposed composite indices for this disease status entity

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Francesca Pignataro ◽  
Wanda Maglione ◽  
Antonina Minniti ◽  
Domenico Sambataro ◽  
Gianluca Sambataro ◽  
...  
Keyword(s):  
Steady State ◽  
Systemic Sclerosis ◽  
Disease Activity ◽  
State Level ◽  
Disease Status ◽  
Cumulative Number ◽  
Nailfold Videocapillaroscopy ◽  
Roc Curve Analysis ◽  
Good Tool ◽  
Steady State Levels

Abstract Background In previous studies, we demonstrated that the NEMO score, i.e. the cumulative number of microhaemorrhages (MHEs) and microthromboses (MTs), observed in nailfold videocapillaroscopy was a good indicator of the steady state level of disease activity (DA) in patients with systemic sclerosis (SSc) when the European Scleroderma Study Group (EScSG) index was considered the gold standard. Aim of the study To verify whether the NEMO score could be (i) a valid tool to assess DA, even when the modified European Scleroderma Trials and Research (EUSTAR) index was considered to be the comparator, and (ii) a sensitive method to capture the DA overtime changes. Patients and methods The NEMO score and the EScSG and EUSTAR indices were contemporarily assessed at baseline (T0) and after a follow-up of 4–56 months (T1) in 98 patients with SSc. The differences (Δ) between the T1 and T0 values of the NEMO score and the EScSG and EUSTAR indices were calculated and compared to each other. Results NEMO score values were very closely correlated with the corresponding values of the EScSG and EUSTAR indices both at T0 and T1 observations (p < 0.0001 in all cases with the exception of the correlation with EScSG values at T1 (p < 0.03)). The values of the two composite DA indices were also strictly related to each other in both T0 and T1 observations (p < 0.0001). Receiver operating characteristic (ROC) curve analysis showed the NEMO score had a good sensitivity and specificity in classifying patients with a predefined level of DA (scores ≥ 3.0 and ≥ 2.5 for the EScSG and EUSTAR indices, respectively, p < 0.0001 in both cases). Δ values of the NEMO score were significantly correlated with the corresponding values of both the EScSG and EUSTAR indices. Weighted Cohen’s k level of agreement between Δ values of the NEMO score and those of the EScSG and EUSTAR indices was moderate (0.55 and 0.59, respectively). Conclusions NEMO score proves to be a feasible, non-invasive, and valid tool to assess steady state levels and changes over time of DA in patients with SSc. Thus, it can represent an alternative or complementary method to measure this disease status entity in this disorder.

Induction of gene expression during involution of the lactating mammary gland of the rat

1994 ◽  
Vol 12 (1) ◽  
pp. 47-60 ◽  
Author(s):  
R S Guenette ◽  
H B Corbeil ◽  
J Léger ◽  
K Wong ◽  
V Mézl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Steady State ◽  
Epithelial Cells ◽  
Tissue Transglutaminase ◽  
State Level ◽  
Ultrastructural Changes ◽  
Steady State Level ◽  
Hsp 27 ◽  
Steady State Levels

ABSTRACT After weaning, the mammary gland ceases lactation and involutes. The wet weight of the gland decreases by 70% within 4 days of weaning. This involves significant tissue remodelling as the ducts regress and return to the resting state. The presence of apoptotic bodies in the luminal epithelial compartment 2 to 3 days after weaning provides clear evidence that a substantial proportion of the regression is attributable to the induction of active cell death (ACD) of the epithelial cells. These changes in the architecture of the gland were found to be mirrored by changes in gene expression. The steady-state level of β-casein mRNA decreased rapidly after weaning from the high levels seen during lactation to undetectable levels by 8 days after weaning. The steady-state levels of expression of a number of genes associated with ACD, including TRPM-2, tissue transglutaminase (TGase) and poly(ADP-ribose) polymerase (PARP), increased transiently during this time-frame. The steady-state level of TRPM-2 mRNA increased 2 days after weaning, reaching a peak on day 4, and decreasing to undetectable levels by day 8 after weaning. The steady-state levels of two other mRNAs, TGase and PARP, showed very similar kinetics. In contrast, the mRNA for Hsp 27, which has been shown to be induced during prostate regression, was not significantly induced in the regressing mammary gland. In-situ hybridization demonstrated that the TRPM-2, TGase and PARP genes were expressed predominantly in the luminal epithelial cells of the ducts. These cells expressed β-casein mRNA during lactation, and underwent ACD after weaning. While the ultrastructural changes in the mammary gland after weaning, and the induction of TRPM-2, TGase and PARP mRNAs, are reminiscent of apoptosis in the prostate, several features of the process are different. Most notably, the disruption of the secretory processes and the lack of increased expression of Hsp 27 in the regressing mammary gland suggest that there may be a number of important events in ACD that are not common to all cells.

scholarly journals Melatonin Mitigates the Infection of Colletotrichum gloeosporioides via Modulation of the Chitinase Gene and Antioxidant Activity in Capsicum annuum L.

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 7
Author(s):  
Muhammad Ali ◽  
Anthony Tumbeh Lamin-Samu ◽  
Izhar Muhammad ◽  
Mohamed Farghal ◽  
Abdul Mateen Khattak ◽  
...  
Keyword(s):  
Antioxidant Enzymes ◽  
Steady State ◽  
Colletotrichum Gloeosporioides ◽  
State Level ◽  
Pathogen Resistance ◽  
Chitinase Gene ◽  
Pathogenesis Related ◽  
Steady State Levels ◽  
Plant Pathogen Interactions ◽  
Pepper Plants

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most damaging pepper (Capsicum annum L.) disease. Melatonin induces transcription of defense-related genes that enhance resistance to pathogens and mediate physiological activities in plants. To study whether the melatonin-mediated pathogen resistance is associated with chitinase gene (CaChiIII2), pepper plants and Arabidopsis seeds were treated with melatonin, then CaChiIII2 activation, hydrogen peroxide (H2O2) levels, and antioxidant enzymes activity during plant–pathogen interactions were investigated. Melatonin pretreatment uncoupled the knockdown of CaChiIII2 and transiently activated its expression level in both control and CaChiIII2-silenced pepper plants and enhanced plant resistance. Suppression of CaChiIII2 in pepper plants showed a significant decreased in the induction of defense-related genes and resistance to pathogens compared with control plants. Moreover, melatonin efficiently enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhanced the activities of antioxidant enzymes, which possibly improved disease resistance. The activation of the chitinase gene CaChiIII2 in transgenic Arabidopsis lines was elevated under C. gloeosporioides infection and exhibited resistance through decreasing H2O2 biosynthesis and maintaining H2O2 at a steady-state level. Whereas melatonin primed CaChiIII2-overexpressed (OE) and wild-type (WT) Arabidopsis seedlings displayed a remarkable increase in root-length compared to the unprimed WT plants. Using an array of CaChiIII2 knockdown and OE, we found that melatonin efficiently induced CaChiIII2 and other pathogenesis-related genes expressions, responsible for the innate immunity response of pepper against anthracnose disease.

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