scholarly journals Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts

2021 ◽  
Vol 22 (10) ◽  
pp. 5293
Author(s):  
Marie Christine Martens ◽  
Janin Edelkamp ◽  
Christina Seebode ◽  
Mirijam Schäfer ◽  
Susanne Stählke ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Human Fibroblasts ◽  
Human Cell Line ◽  
Epidermal Differentiation ◽  
Loss Of Function ◽  
Lentiviral Transduction ◽  
Protein Receptor ◽  
Models Comparison

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

scholarly journals The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome

2011 ◽  
Vol 22 (24) ◽  
pp. 4776-4786 ◽  
Author(s):  
Gulistan Mese ◽  
Caterina Sellitto ◽  
Leping Li ◽  
Hong-Zhan Wang ◽  
Virginijus Valiunas ◽  
...  
Keyword(s):  
Animal Model ◽  
Mouse Model ◽  
Transgenic Mouse ◽  
Hair Loss ◽  
Epidermal Differentiation ◽  
Gjb2 Gene ◽  
Loss Of Function ◽  
Nonsyndromic Deafness ◽  
Connexin Hemichannels

Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders.

scholarly journals Genome-Wide Identification and Characterization of MLO Gene Family in Octoploid Strawberry (Fragaria ×ananassa)

2020 ◽  
Author(s):  
Ronald R. Tapia ◽  
Christopher R. Barbey ◽  
Saket Chandra ◽  
Kevin M. Folta ◽  
Vance M. Whitaker ◽  
...  
Keyword(s):  
Gene Family ◽  
Genomic Structure ◽  
Resistance Locus ◽  
Fragaria Ananassa ◽  
Loss Of Function ◽  
Genetic Studies ◽  
Genome Wide ◽  
Segregating Population

AbstractPowdery mildew (PM) caused by Podosphaera aphanis is a major fungal disease in cultivated strawberry. Mildew Resistance Locus O (MLO) is a gene family described for having conserved seven-transmembrane domains. Induced loss-of-function in specific MLO genes can confer durable and broad resistance against PM pathogens. However, the underlying biological role of MLO genes in strawberry is still unknown. In the present study, the genomic structure of MLO genes were characterized in both diploid (Fragaria vesca) and octoploid strawberry (Fragaria ×ananassa), and the potential sources of MLO-mediated susceptibility were identified. Twenty MLO-like sequences were identified in F. vesca, with sixty-eight in F. ×ananassa. Phylogenetic analysis divides strawberry MLO genes into eight different clades, in which three FveMLO and ten FaMLO genes were grouped together with the functionally known MLO susceptibility. Out of ten FaMLO genes, FaMLO17-2 and FaMLO17-3 showed the highest similarity to the known susceptibility MLO proteins. Gene expression analysis of FaMLO genes was conducted using a multi-parental segregating population. Three expression quantitative trait loci (eQTL) were substantially associated with MLO transcript levels in mature fruits, suggesting discrete genetic control of susceptibility. These results are a critical first step in understanding allele function of MLO genes, and are necessary for further genetic studies of PM resistance in cultivated strawberry.

scholarly journals Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases

2018 ◽  
Vol 116 (3) ◽  
pp. 970-975 ◽  
Author(s):  
Yue Li ◽  
Marita Führer ◽  
Ehsan Bahrami ◽  
Piotr Socha ◽  
Maja Klaudel-Dreszler ◽  
...  
Keyword(s):  
Cell Death ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Loss Of Function ◽  
Monogenic Disorders ◽  
Bowel Diseases ◽  
Inflammatory Bowel ◽  
Human Immune

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

scholarly journals Characterization of the Endogenous DAF-12 Ligand and Its Use as an Anthelmintic Agent in Strongyloides stercoralis

2021 ◽  
Author(s):  
Zhu Wang ◽  
Mi Cheong Cheong ◽  
Jet Tsien ◽  
Heping Deng ◽  
Tian Qin ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Strongyloides Stercoralis ◽  
Preclinical Model ◽  
Loss Of Function ◽  
Drug Of Choice ◽  
Lethal Infection ◽  
Parasite Reproduction ◽  
Rate Limiting

ABSTRACTA prevalent feature of Strongyloides stercoralis is a life-long and potentially lethal infection that is due to the nematode parasite’s ability to autoinfect and, thereby, self-replicate within its host. Here, we investigated the role of the parasite’s nuclear receptor, Ss-DAF-12, in governing infection. We identified Δ7-DA as the endogenous Ss-DAF-12 ligand and elucidated the hormone’s biosynthetic pathway. Genetic loss of function of the ligand’s rate-limiting enzyme demonstrated that Δ7-DA synthesis is necessary for parasite reproduction, whereas its absence is required for development of infectious larvae. Availability of the ligand permits Ss-DAF-12 to function as an on/off switch governing autoinfection, making it vulnerable to therapeutic intervention. In a preclinical model of hyperinfection, pharmacologic activation of DAF-12 suppressed autoinfection and markedly reduced lethality. Moreover, when Δ7-DA was administered with ivermectin, the current but limited drug of choice for treating strongyloidiasis, the combinatorial effects of the two drugs resulted in a near cure of the disease.

scholarly journals Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins

1999 ◽  
Vol 19 (11) ◽  
pp. 7870-7885 ◽  
Author(s):  
Nieves Movilla ◽  
Xosé R. Bustelo
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Wild Type ◽  
Gtp Binding ◽  
Regulatory Properties ◽  
Identification And Characterization

ABSTRACT We report here the identification and characterization of a novel Vav family member, Vav-3. Signaling experiments demonstrate that Vav-3 participates in pathways activated by protein tyrosine kinases. Vav-3 promotes the exchange of nucleotides on RhoA, on RhoG and, to a lesser extent, on Rac-1. During this reaction, Vav-3 binds physically to the nucleotide-free states of those GTPases. These functions are stimulated by tyrosine phosphorylation in wild-type Vav-3 and become constitutively activated upon deletion of the entire calponin-homology region. Expression of truncated versions of Vav-3 leads to drastic actin relocalization and to the induction of stress fibers, lamellipodia, and membrane ruffles. Moreover, expression of Vav-3 alters cytokinesis, resulting in the formation of binucleated cells. All of these responses need only the expression of the central region of Vav-3 encompassing the Dbl homology (DH), pleckstrin homology (PH), and zinc finger (ZF) domains but do not require the presence of the C-terminal SH3-SH2-SH3 regions. Studies conducted with Vav-3 proteins containing loss-of-function mutations in the DH, PH, and ZF regions indicate that only the DH and ZF regions are essential for Vav-3 biological activity. Finally, we show that one of the functions of the Vav-3 ZF region is to work coordinately with the catalytic DH region to promote both the binding to GTP-hydrolases and their GDP-GTP nucleotide exchange. These results highlight the role of Vav-3 in signaling and cytoskeletal pathways and identify a novel functional cross-talk between the DH and ZF domains of Vav proteins that is imperative for the binding to, and activation of, Rho GTP-binding proteins.

scholarly journals Twitchy, the Drosophila orthologue of the ciliary gating protein FBF1/dyf-19, is required for coordinated locomotion and male fertility

Biology Open ◽  
2021 ◽  
Author(s):  
Suzanne H Hodge ◽  
Amy Watts ◽  
Richard Marley ◽  
Richard A Baines ◽  
Ernst Hafen ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Primary Cilia ◽  
Loss Of Function ◽  
Transmembrane Receptors ◽  
C Elegans ◽  
Male Germline ◽  
Sensory Cilia ◽  
Import And Export ◽  
Flagellar Axoneme

Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. These are required for the assembly of the cilium, its function as a sensory and signalling centre and to maintain its distinctive composition. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalized primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. Here, we describe phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. Twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalized from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells.

scholarly journals Functional characterization of ribosomal P1/P2 proteins in human cells

2008 ◽  
Vol 413 (3) ◽  
pp. 527-534 ◽  
Author(s):  
Francisco Martinez-Azorin ◽  
Miguel Remacha ◽  
Juan P. G. Ballesta
Keyword(s):  
Functional Characterization ◽  
Ribosomal Subunit ◽  
Human Cell Line ◽  
Human Cells ◽  
Translation Efficiency ◽  
Ribosomal Stalk ◽  
P Proteins ◽  
Subunit Joining

The ‘stalk’ is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.

Differential expression of two cadherins in Xenopus laevis

Development ◽  
1991 ◽  
Vol 111 (3) ◽  
pp. 829-844 ◽  
Author(s):  
B. Angres ◽  
A.H. Muller ◽  
J. Kellermann ◽  
P. Hausen
Keyword(s):  
Neural Induction ◽  
Epidermal Differentiation ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Cell Layers ◽  
Plate Area ◽  
Apical Membranes ◽  
Embryonic Ectoderm

Using a cadherin fraction from Xenopus tissue culture cells as an immunogen, two monoclonal antibodies were obtained that allowed the characterization of two distinct cadherins in the Xenopus embryo. The two cadherins differ in molecular weight, in their time of appearance during development and in their spatial pattern of expression. One of the antigens was identified as E-cadherin. It appears in the embryonic ectoderm during gastrulation when epidermal differentiation commences and it disappears from the neural plate area upon neural induction. The second antigen could not be allocated to any of the known cadherin subtypes and was termed U-cadherin. It is present in the egg and becomes deposited in newly formed inner cell membranes during cleavage, the outer apical membranes of the embryo remaining devoid of the cadherin throughout development. U-cadherin is found on membranes of all cells up to the late neurula stages. A conspicuous polarized expression of the antigen on the membranes of individual inner cells suggests its participation in the segregation of cell layers and organ anlagen. These findings are discussed in the context of current hypotheses on the role of cadherins in establishing the spatial structure of the embryo.

scholarly journals Characterization of the endogenous DAF-12 ligand and its use as an anthelmintic agent in Strongyloides stercoralis

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Zhu Wang ◽  
Mi Cheong Cheong ◽  
Jet Tsien ◽  
Heping Deng ◽  
Tian Qin ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Strongyloides Stercoralis ◽  
Preclinical Model ◽  
Loss Of Function ◽  
Drug Of Choice ◽  
Lethal Infection ◽  
Parasite Reproduction ◽  
Rate Limiting

A prevalent feature of Strongyloides stercoralis is a life-long and potentially lethal infection that is due to the nematode parasite’s ability to autoinfect and, thereby, self-replicate within its host. Here, we investigated the role of the parasite’s nuclear receptor, Ss-DAF-12, in governing infection. We identified Δ7-DA as the endogenous Ss-DAF-12 ligand and elucidated the hormone’s biosynthetic pathway. Genetic loss of function of the ligand’s rate-limiting enzyme demonstrated that Δ7-DA synthesis is necessary for parasite reproduction, whereas its absence is required for the development of infectious larvae. Availability of the ligand permits Ss-DAF-12 to function as an on/off switch governing autoinfection, making it vulnerable to therapeutic intervention. In a preclinical model of hyperinfection, pharmacologic activation of DAF-12 suppressed autoinfection and markedly reduced lethality. Moreover, when Δ7-DA was administered with ivermectin, the current but limited drug of choice for treating strongyloidiasis, the combinatorial effects of the two drugs resulted in a near cure of the disease.

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