scholarly journals Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2736-2742 ◽  
Author(s):  
Gaël Ménasché ◽  
Jérôme Feldmann ◽  
Anne Houdusse ◽  
Catherine Desaymard ◽  
Alain Fischer ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Nucleotide Binding ◽  
Binding Activity ◽  
Small Gtpase ◽  
Rab Proteins ◽  
Missense Mutations ◽  
Immune Disorder ◽  
Griscelli Syndrome ◽  
Protein Functions ◽  
Cytotoxic Granule

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the α4 (Ala152Pro) or β5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

scholarly journals A Phytophthora capsici RXLR effector manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein trafficking pathway

2021 ◽  
Author(s):  
Tianli Li ◽  
Gan Ai ◽  
Xiaowei Fu ◽  
Jin Liu ◽  
Hai Zhu ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Plant Immunity ◽  
Phytophthora Capsici ◽  
Functional Characterization ◽  
Ectopic Expression ◽  
Defense Responses ◽  
Infection Process ◽  
Small Gtpase ◽  
Rab Proteins ◽  
Rxlr Effector

The oomycete pathogen Phytophthora capsici encodes hundreds of RXLR effectors to enter plant cells and suppress host defense responses. Only few of them are conserved across different strains and species. Such ‘core effectors’ may target hub immunity pathways that are essential during Phytophthora pathogens interacting with their hosts. However, the underlying mechanisms of core RXLRs-mediated host immunity manipulation are largely unknown. Here, we report the functional characterization of a P. capsici RXLR effector, RXLR242. RXLR242 expression is highly induced during the infection process. Its ectopic expression in Nicotiana benthamiana promotes Phytophthora infection. RXLR242 physically interacts with a group of RAB proteins, which belong to the small GTPase family and function in specifying transport pathways in the intracellular membrane trafficking system. RXLR242 impedes the secretion of PATHOGENESIS-RELATED 1 (PR1) protein to the apoplast by interfering the formation of RABE1-7-labeled vesicles. Further analysis indicated that such phenomenon is resulted from competitive binding of RXLR242 to RABE1-7. RXLR242 also interferes trafficking of the membrane-located receptor FLAGELLIN-SENSING 2 (FLS2) through competitively interacting with RABA4-3. Taken together, our work demonstrates that RXLR242 manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein transporting pathway in plant hosts.

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

2010 ◽  
Vol 23 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Norihiko Ohbayashi ◽  
Setareh Mamishi ◽  
Koutaro Ishibashi ◽  
Yuto Maruta ◽  
Babak Pourakbari ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Syndrome Type ◽  
Missense Mutations ◽  
Griscelli Syndrome

scholarly journals Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

2021 ◽  
Vol 22 (5) ◽  
pp. 2732
Author(s):  
Nadine Reichhart ◽  
Vladimir M. Milenkovic ◽  
Christian H. Wetzel ◽  
Olaf Strauß
Keyword(s):  
Transmembrane Protein ◽  
Functional Characterization ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Functional Consequences ◽  
New Perspective ◽  
The Stability ◽  
Dynamics Simulations ◽  
And Function

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

Oxidative Modification of Fibrinogen Affects Its Binding Activity to Glycoprotein (GP) IIb/IIIa

2009 ◽  
Vol 16 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Sermin Tetik ◽  
Kurtulus Kaya ◽  
M. Demir ◽  
Emel Eksioglu-Demiralp ◽  
Turay Yardimci
Keyword(s):  
Metal Ion ◽  
Degradation Products ◽  
Binding Capacity ◽  
Protein Structures ◽  
Human Platelets ◽  
Binding Activity ◽  
Oxidative Modification ◽  
Oxidized Fibrinogen ◽  
Protein Functions

Aim: Proteins are sensitive biomarkers of human diease condition associated with oxidative stress. Alteration of protein structures by oxidants may result in partial or complete loss of protein functions. We have investigated the effect of structural modifications induced by metal ion catalyzed oxidation of fibrinogen on its binding capacity to glycoprotein IIb/IIIa (GpIIb/IIIa) and human platelets. Methods: We identified and quantified of binding capacity of native and oxidized fibrinogen to its receptor in vitro by flow cytometer. Dityrosine formation on oxidized fibrinogen were detected spectrophotometrically. Elevated degradation products of fibrinogen after oxidation were revealed in the HPLC analysis. The native and oxidized fibrinogen were analyzed on mass spectrum upon digestion with tyripsin. Results: Oxidatively modified fibrinogen showed less binding activity than native fibrinogen to GpIIb/IIIa coated micro beads and human platelets whereas slightly higher binding capaticity to ADP induced stimulated platelets. Formation of dityrosines in the amino acid side chains of fibrinogen were observed upon oxidation. Decreased binding capacity of oxidized fibrinogen correlated with intensities of dityrosine formation. Oxidized fibrinogen had more ion-mass intensities at higher than native fibrinogen. Clinical implications: Important point is decreased of binding capacity of the oxidized fibrinogen to own receptor. The decreased rate of binding, leading to effect in the diseases of clot formation may acount for the association between oxidation of fibrinogen and the incidence of effect in human diseases.

The role of host cell Rab GTPases in influenza A virus infections

2021 ◽  
Author(s):  
Jing Wu ◽  
Jiaqi Gu ◽  
Li Shen ◽  
Xiaonan Jia ◽  
Yiqian Yin ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Respiratory Infections ◽  
Small Gtpase ◽  
Regulatory Mechanisms ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Virus Infections ◽  
Adaptation Mechanisms

Influenza A virus (IAV) is a crucial cause of respiratory infections in humans worldwide. Therefore, studies should clarify adaptation mechanisms of IAV and critical factors of the viral pathogenesis in human hosts. GTPases of the Rab family are the largest branch of the Ras-like small GTPase superfamily, and they regulate almost every step during vesicle-mediated trafficking. Evidence has shown that Rab proteins participate in the lifecycle of IAV. In this mini-review, we outline the regulatory mechanisms of different Rab proteins in the lifecycle of IAV. Understanding the role of Rab proteins in IAV infections is important to develop broad-spectrum host-targeted antiviral strategies.

scholarly journals Functional Characterization of a Novel Promoter Element Required for an Innate Immune Response in Drosophila

2003 ◽  
Vol 23 (22) ◽  
pp. 8272-8281 ◽  
Author(s):  
Hanna Uvell ◽  
Ylva Engström
Keyword(s):  
Gene Expression ◽  
Antimicrobial Peptide ◽  
Functional Characterization ◽  
Binding Activity ◽  
Innate Immune ◽  
Site Directed Mutagenesis ◽  
Promoter Element ◽  
Peptide Gene ◽  
Inducible Genes

ABSTRACT Innate immune reactions are crucial processes of metazoans to protect the organism against overgrowth of faster replicating microorganisms. Drosophila melanogaster is a precious model for genetic and molecular studies of the innate immune system. In response to infection, the concerted action of a battery of antimicrobial peptides ensures efficient killing of the microbes. The induced gene expression relies on translocation of the Drosophila Rel transcription factors Relish, Dif, and Dorsal to the nucleus where they bind to κB-like motifs in the promoters of the inducible genes. We have identified another putative promoter element, called region 1 (R1), in a number of antimicrobial peptide genes. Site-directed mutagenesis of the R1 site diminished Cecropin A1 (CecA1) expression in transgenic Drosophila larvae and flies. Infection of flies induced a nuclear R1-binding activity that was unrelated to the κB-binding activity in the same extracts. Although the R1 motif was required for Rel protein-mediated CecA1 expression in cotransfection experiments, our data argue against it being a direct target for the Drosophila Rel proteins. We propose that the R1 and κB motifs are targets for distinct regulatory complexes that act in concert to promote high levels of antimicrobial peptide gene expression in response to infection.

Monogenic Epilepsies: Disease Mechanisms, Clinical Phenotypes, and Targeted Therapies

Neurology ◽  
2021 ◽  
pp. 10.1212/WNL.0000000000012744
Author(s):  
Renzo Guerrini ◽  
Simona Balestrini ◽  
Elaine C. Wirrell ◽  
Matthew C. Walker
Keyword(s):  
Observational Studies ◽  
Current Knowledge ◽  
Functional Characterization ◽  
Treatment Strategies ◽  
Controlled Trials ◽  
Epileptogenic Zone ◽  
Missense Mutations ◽  
Number Of Patients ◽  
Functional Consequences ◽  
Genetic Epilepsies

A monogenic aetiology can be identified in up to 40% of people with severe epilepsy. To address earlier and more appropriate treatment strategies, clinicians are required to know the implications that specific genetic causes might have on pathophysiology, natural history, comorbidities and treatment choices. In this narrative review, we summarise concepts on the genetic epilepsies based on the underlying pathophysiological mechanisms and present the current knowledge on treatment options based on evidence provided by controlled trials or studies with lower classification of evidence. Overall, evidence robust enough to guide antiseizure medication (ASM) choices in genetic epilepsies remains limited to the more frequent conditions for which controlled trials and observational studies have been possible. Most monogenic disorders are very rare and ASM choices for them are still based on inferences drawn from observational studies and early, often anecdotal, experiences with precision therapies. Precision medicine remains applicable to only a narrow number of patients with monogenic epilepsies and may target only part of the actual functional defects. Phenotypic heterogeneity is remarkable, and some genetic mutations activate epileptogenesis through their developmental effects, which may not be reversed postnatally. Other genes seem to have pure functional consequences on excitability, acting through either loss- or gain-of-function effects, and these may have opposite treatment implications. In addition, the functional consequences of missense mutations may be difficult to predict, making precision treatment approaches considerably more complex than estimated by deterministic interpretations. Knowledge of genetic aetiologies can influence the approach to surgical treatment of focal epilepsies. Identification of germline mutations in specific genes contraindicates surgery while mutations in other genes do not. Identification, quantification and functional characterization of specific somatic mutations before surgery using cerebrospinal fluid liquid biopsy or after surgery in brain specimens, will likely be integrated in planning surgical strategies and re-intervention after a first unsuccessful surgery as initial evidence suggests that mutational load may correlate with the epileptogenic zone. Promising future directions include gene manipulation by DNA or mRNA targeting; although most are still far from clinical use, some are in early phase clinical development.

Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP

1991 ◽  
Vol 11 (9) ◽  
pp. 4717-4725
Author(s):  
Y S Cheng ◽  
C E Patterson ◽  
P Staeheli
Keyword(s):  
Binding Proteins ◽  
Nucleotide Binding ◽  
Binding Activity ◽  
Guanine Nucleotides ◽  
Sequence Motifs ◽  
Consensus Motif ◽  
Gtp Binding ◽  
Genes Encoding ◽  
Nucleotide Binding Proteins ◽  
High Degree

The primary structures of interferon (IFN)-induced guanylate-binding proteins (GBPs) were deduced from cloned human and murine cDNAs. These proteins contained only two of the three sequence motifs typically found in GTP/GDP-binding proteins. The N(T)KXD motif, which is believed to confer guanine specificity in other nucleotide-binding proteins, was absent. Nevertheless, the IFN-induced GBPs exhibited a high degree of selectivity for binding to agarose-immobilized guanine nucleotides. An interesting feature of IFN-induced GBPs is that they strongly bound to GMP agarose in addition to GDP and GTP agaroses but failed to bind to ATP agarose and all other nucleotide agaroses tested. Both GTP and GMP, but not ATP, competed for binding of murine GBP-1 to agarose-immobilized GMP. The IFN-induced GBPs thus define a distinct novel family of proteins with GTP-binding activity. We further demonstrate that human and murine cells contain at least two genes encoding IFN-induced GBPs. The cloned murine cDNA codes for GBP-1, an IFN-induced protein previously shown to be absent from mice of Gbp-1b genotype.

A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5052-5062 ◽  
Author(s):  
Gaël Ménasché ◽  
Mickaël M. Ménager ◽  
Juliette M. Lefebvre ◽  
Einat Deutsch ◽  
Rafika Athman ◽  
...  
Keyword(s):  
Target Cell ◽  
Affinity Purification ◽  
Cytotoxic T Cells ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Granule Secretion ◽  
Immunologic Synapse ◽  
Cytotoxic Granule

Abstract Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules—indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

scholarly journals Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension

1996 ◽  
Vol 271 (4) ◽  
pp. C1172-C1180 ◽  
Author(s):  
B. H. Jiang ◽  
G. L. Semenza ◽  
C. Bauer ◽  
H. H. Marti
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Hypoxia Inducible Factor ◽  
Binding Activity ◽  
Maximal Response ◽  
Hypoxia Inducible Factor 1 ◽  
Dna Binding Activity

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein implicated in the transcriptional activation of genes encoding erythropoietin, glycolytic enzymes, and vascular endothelial growth factor in hypoxic mammalian cells. In this study, we have quantitated HIF-1 DNA-binding activity and protein levels of the HIF-1 alpha and HIF-1 beta subunits in human HeLa cells exposed to O2 concentrations ranging from 0 to 20% in the absence or presence of 1 mM KCN to inhibit oxidative phosphorylation and cellular O2 consumption. HIF-1 DNA-binding activity, HIF-1 alpha protein and HIF-1 beta protein each increased exponentially as cells were subjected to decreasing O2 concentrations, with a half maximal response between 1.5 and 2% O2 and a maximal response at 0.5% O2, both in the presence and absence of KCN. The HIF-1 response was greatest over O2 concentrations associated with ischemic/hypoxic events in vivo. These results provide evidence for the involvement of HIF-1 in O2 homeostasis and represent a functional characterization of the putative O2 sensor that initiates hypoxia signal transduction leading to HIF-1 expression.

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