scholarly journals Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jamie C Little ◽  
Elisa Garcia-Garcia ◽  
Amanda Sul ◽  
Daniel Kalderon
Keyword(s):  
Target Gene ◽  
Gene Activation ◽  
Proteolytic Processing ◽  
Full Length ◽  
Target Cells ◽  
Transcriptional Repressors ◽  
Gli Proteins ◽  
Transcriptional Changes ◽  
Wing Imaginal Discs ◽  
Dose Dependent

Extracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length Ci or Gli proteins. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling in Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology, when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, likely acting through binding the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci processing.

scholarly journals Drosophila Hedgehog can act as a morphogen in the absence of regulated Ci processing

2020 ◽  
Author(s):  
Jamie C. Little ◽  
Elisa Garcia-Garcia ◽  
Amanda Sul ◽  
Daniel Kalderon
Keyword(s):  
Protein Profile ◽  
Gene Activation ◽  
Proteolytic Processing ◽  
Full Length ◽  
Target Cells ◽  
Linear Gradient ◽  
Gli Proteins ◽  
Transcriptional Changes ◽  
Wing Imaginal Discs ◽  
Dose Dependent

SummaryExtracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length proteins, which are otherwise held inactive by cytoplasmic binding partners and subject to accelerated degradation following activation. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling at the anteroposterior (AP) border of Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The full-length Ci-155 protein profile of these variants revealed a linear gradient of Hh-stimulated degradation, allowing derivation of a spatial profile of inhibition of processing of normal C-155 by Hh. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, acting through association with the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci-155 processing.

scholarly journals Sites and Determinants of Early Cleavages in the Proteolytic Processing Pathway of Reovirus Surface Protein σ3

2002 ◽  
Vol 76 (10) ◽  
pp. 5184-5197 ◽  
Author(s):  
Judit Jané-Valbuena ◽  
Laura A. Breun ◽  
Leslie A. Schiff ◽  
Max L. Nibert
Keyword(s):  
Structural Changes ◽  
Proper Time ◽  
Strain Differences ◽  
Surface Protein ◽  
Cellular Membrane ◽  
Proteolytic Processing ◽  
Target Cells ◽  
Inactive Form ◽  
Reovirus Type ◽  
Type 3

ABSTRACT Entry of mammalian reovirus virions into target cells requires proteolytic processing of surface protein σ3. In the virion, σ3 mostly covers the membrane-penetration protein μ1, appearing to keep it in an inactive form and to prevent it from interacting with the cellular membrane until the proper time in infection. The molecular mechanism by which σ3 maintains μ1 in this inactive state and the structural changes that accompany σ3 processing and μ1 activation, however, are not well understood. In this study we characterized the early steps in σ3 processing and determined their effects on μ1 function and particle infectivity. We identified two regions of high protease sensitivity, “hypersensitive” regions located at residues 208 to 214 and 238 to 244, within which all proteases tested selectively cleaved σ3 as an early step in processing. Further processing of σ3 was required for infection, consistent with the fact that the fragments resulting from these early cleavages remained bound to the particles. Reovirus type 1 Lang (T1L), type 3 Dearing (T3D), and T1L × T3D reassortant virions differed in the sites of early σ3 cleavage, with T1L σ3 being cleaved mainly at residues 238 to 244 and T3D σ3 being cleaved mainly at residues 208 to 214. These virions also differed in the rates at which the early cleavages occurred, with cleavage of T1L σ3 occurring faster than cleavage of T3D σ3. Analyses using chimeric and site-directed mutants of recombinant σ3 identified carboxy-proximal residues 344, 347, and 353 as the primary determinants of these strain differences. The spatial relationships between these more carboxy-proximal residues and the hypersensitive regions were discerned from the σ3 crystal structure. The results indicate that proteolytic processing of σ3 during reovirus disassembly is a multistep pathway with a number of molecular determinants.

Progesterone enhances target gene transcription by receptor free of heat shock proteins hsp90, hsp56, and hsp70

1991 ◽  
Vol 11 (10) ◽  
pp. 4998-5004
Author(s):  
M K Bagchi ◽  
S Y Tsai ◽  
M J Tsai ◽  
B W O'Malley
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Heat Shock Protein ◽  
Transcriptional Activation ◽  
Target Gene ◽  
Steroid Hormone ◽  
Receptor Activation ◽  
Target Cells ◽  
Dependent Manner ◽  
Shock Proteins

Steroid receptors regulate transcription of target genes in vivo and in vitro in a steroid hormone-dependent manner. Unoccupied progesterone receptor exists in the low-salt homogenates of target cells as a functionally inactive 8 to 10S complex with several nonreceptor components such as two molecules of 90-kDa heat shock protein (hsp90), a 70-kDa heat shock protein (hsp70), and a 56-kDa heat shock protein (hsp56). Ligand-induced dissociation of receptor-associated proteins such as hsp90 has been proposed as the mechanism of receptor activation. Nevertheless, it has not been established whether, beyond release of heat shock proteins, the steroidal ligand plays a role in modulating receptor activity. To examine whether the release of these nonreceptor proteins from receptor complex results in a constitutively active receptor, we isolated an unliganded receptor form essentially free of hsp90, hsp70, and hsp56. Using a recently developed steroid hormone-responsive cell-free transcription system, we demonstrate for the first time that the dissociation of heat shock proteins is not sufficient to generate a functionally active receptor. This purified receptor still requires hormone for high-affinity binding to a progesterone response element and for efficient transcriptional activation of a target gene. When an antiprogestin, Ru486, is bound to the receptor, it fails to promote efficient transcription. We propose that in the cell, in addition to the release of receptor-associated inhibitory proteins, a distinct hormone-mediated activation event must precede efficient gene activation.

scholarly journals FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

Blood ◽  
2019 ◽  
Vol 133 (8) ◽  
pp. 830-839 ◽  
Author(s):  
Viola Close ◽  
William Close ◽  
Sabrina Julia Kugler ◽  
Michaela Reichenzeller ◽  
Deyan Yordanov Yosifov ◽  
...  
Keyword(s):  
Target Gene ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Missense Mutations ◽  
Protein Levels ◽  
In Silico Modeling ◽  
Functional Consequences ◽  
Wd40 Domain

Abstract NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Dose dependent transcriptional changes in circulating myeloid cells following chronic ethanol consumption

Alcohol ◽  
2018 ◽  
Vol 66 ◽  
pp. 95 ◽  
Author(s):  
S. Sureshchandra ◽  
A. Jankeel ◽  
C. Stull ◽  
K. Grant ◽  
Ilhem Messaoudi
Keyword(s):  
Myeloid Cells ◽  
Ethanol Consumption ◽  
Chronic Ethanol ◽  
Chronic Ethanol Consumption ◽  
Transcriptional Changes ◽  
Dose Dependent

scholarly journals Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1166 ◽  
Author(s):  
Olivia R. Buonarati ◽  
Peter B. Henderson ◽  
Geoffrey G. Murphy ◽  
Mary C. Horne ◽  
Johannes W. Hell
Keyword(s):  
Gene Expression ◽  
Brain Tissue ◽  
Neuronal Excitability ◽  
Central Element ◽  
Proteolytic Processing ◽  
Full Length ◽  
Hek293 Cells ◽  
Specific Antibodies ◽  
C Terminus ◽  
Molecular Masses

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (MR)of the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.

Abstract P191: Calpain-Generated Free PKCα Catalytic Domains Induce HDAC5 Nuclear Export and Regulate Cardiac Gene Transcription

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Yan Zhang ◽  
Scot Matkovich ◽  
Abhinav Diwan ◽  
Min-Young Kang ◽  
Gerald W Dorn
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Nuclear Export ◽  
Kinase Inhibitor ◽  
Proteolytic Processing ◽  
Full Length ◽  
Independent Effect ◽  
Independent Manner ◽  
Cardiac Gene

Receptor-mediated activation of protein kinase (PK) C is a central pathway regulating cell growth, homeostasis, and programmed death. Recently, we showed that calpain-mediated proteolytic processing of PKC in ischemic myocardium activates PKC signaling in a receptor-independent manner by releasing a persistent and constitutively active free catalytic C-terminal fragment, PKCα-CT. This unregulated kinase provokes cardiomyopathy, but the mechanisms remain unclear. We examined hypothesis that PKCα-CT has transcriptional activity. Using immunoblot analysis and confocal microscopy, we found that PKCα-CT localized in part to nuclei and spontaneously induced cytosolic relocalization HDAC5 of the transcriptional regulator. Co- expression of calpain 1 with full length PKCα can generate PKCα-CT and produced the same HDAC5 cytosolic relocalization, whereas full length PKCα alone had no such effect. HDAC5 cytosolic relocalization induced by PKCα-CT was abolished by the protein kinase inhibitor GO6976, but not by PKD inhibitor CID 755673. The in vivo relevance of these findings was examined in transgenic mice expressing PKCα and PKCα-CT. To assess the consequence on gene expression, we performed global transcriptome profiling by Affymetrix microarrays and mRNA sequencing. The two techniques substantially agreed. Compared to control hearts, 621 mRNAs were regulated at least 1.3 fold in PKCα-CT hearts (P< 0.001), only 59 in full-length PKCα hearts. MEF2-dependent inflammatory pathway genes which are putative HDAC targets were upregulated in PKCα-CT heart: 15 MEF2 target mRNAs were upregulated in PKCα-CT hearts (p<0.001), only one in PKCα hearts. These results reveal that PKCα-CT is a potent regulator of pathological cardiac gene expression by localizing to nuclei and directly promoting nuclei-cytoplasmic shuttling of HDAC5. Receptor-independent effect of PKCα-CT and HDAC phosphorylation in ischemic hearts has broad ramifications for understanding and preventing the pathological transcriptional stress response.

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