scholarly journals The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development

2019 ◽  
Vol 29 (2) ◽  
pp. 248-263 ◽  
Author(s):  
Belal Shohayeb ◽  
Uda Ho ◽  
Yvonne Y Yeap ◽  
Robert G Parton ◽  
S Sean Millard ◽  
...  
Keyword(s):  
Radial Glia ◽  
Single Amino Acid ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Protein Loss ◽  
Mutant Proteins ◽  
Cilia Formation ◽  
Morphological Defects ◽  
Cell Processes ◽  
Radial Glial

Abstract WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.

scholarly journals Loss-of-Function Piezo1 Mutations Display Altered Stability Driven by Ubiquitination and Proteasomal Degradation

2021 ◽  
Vol 12 ◽  
Author(s):  
Zijing Zhou ◽  
Jinyuan Vero Li ◽  
Boris Martinac ◽  
Charles D. Cox
Keyword(s):  
Membrane Trafficking ◽  
Proteasome Inhibition ◽  
Proteasomal Degradation ◽  
Missense Mutations ◽  
Turnover Rates ◽  
Loss Of Function ◽  
Wild Type ◽  
Mutant Proteins ◽  
Lymphatic Dysplasia ◽  
Therapeutic Avenue

Missense mutations in the gene that encodes for the mechanically-gated ion channel Piezo1 have been linked to a number of diseases. Gain-of-function variants are linked to a hereditary anaemia and loss-of-function variants have been linked to generalized lymphatic dysplasia and bicuspid aortic valve. Two previously characterized mutations, S217L and G2029R, both exhibit reduced plasma membrane trafficking. Here we show that both mutations also display reduced stability and higher turnover rates than wild-type Piezo1 channels. This occurs through increased ubiquitination and subsequent proteasomal degradation. Congruent with this, proteasome inhibition using N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) reduced the degradation of both mutant proteins. While ALLN treatment could not rescue the function of S217L we show via multiple complementary methodologies that proteasome inhibition via ALLN treatment can not only prevent G2029R turnover but increase the membrane localized pool of this variant and the functional Piezo1 mechanosensitive currents. This data in combination with a precision medicine approach provides a new potential therapeutic avenue for the treatment of Piezo1 mediated channelopathies.

scholarly journals Disease-associated missense mutations in the EVH1 domain disrupt intrinsic WASp function causing dysregulated actin dynamics and impaired dendritic cell migration

Blood ◽  
2013 ◽  
Vol 121 (1) ◽  
pp. 72-84 ◽  
Author(s):  
Austen J. J. Worth ◽  
Joao Metelo ◽  
Gerben Bouma ◽  
Dale Moulding ◽  
Marco Fritzsche ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Actin Dynamics ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Interacting Protein ◽  
Mutant Proteins ◽  
Dendritic Cell Migration ◽  
Biologic Function

Abstract Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.

scholarly journals Isolation and Characterization of Nonchemotactic CheZ Mutants of Escherichia coli

2000 ◽  
Vol 182 (12) ◽  
pp. 3544-3552 ◽  
Author(s):  
Kristin C. Boesch ◽  
Ruth E. Silversmith ◽  
Robert B. Bourret
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Random Mutagenesis ◽  
Signal Transduction Pathway ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Functional Impairments ◽  
Biophysical Characterization ◽  
Mutant Proteins ◽  
Isolation And Characterization

ABSTRACT The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ1–189 peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.

scholarly journals A SMAD1/5-YAP signaling module drives radial glia self-amplification and growth of the developing cerebral cortex

2019 ◽  
Author(s):  
Sonia Najas ◽  
Isabel Pijuan ◽  
Anna Esteve-Codina ◽  
Susana Usieto ◽  
Juan D. Martinez ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Cortical Neurons ◽  
Radial Glia ◽  
Hippo Signaling ◽  
Loss Of Function ◽  
Radial Glial Cells ◽  
Evolutionarily Conserved ◽  
Radial Glial ◽  
Early Mouse ◽  
Post Transcriptional Regulation

AbstractThe growth and evolutionary expansion of the cerebral cortex are defined by the spatial-temporal production of neurons, which itself depends on the decision of radial glial cells (RGCs) to self-amplify or to switch to neurogenic divisions. The mechanisms regulating these RGC fate decisions are still incompletely understood. Here we describe a novel and evolutionarily conserved role of the canonical BMP transcription factors SMAD1/5 in controlling neurogenesis and growth during corticogenesis. Reducing the expression of both SMAD1 and SMAD5 in neural progenitors at early mouse cortical development caused microcephaly and an increased production of early-born cortical neurons at the expense of late-born ones, which correlated with the premature differentiation and depletion of the pool of cortical progenitors. Gain- and loss-of-function experiments performed during early cortical neurogenesis in the chick revealed that SMAD1/5 activity supports self-amplifying RGC divisions and restrain the neurogenic ones. Furthermore, we demonstrate that SMAD1/5 stimulate RGC self-amplification through the positive post-transcriptional regulation of the Hippo signaling effector YAP. We anticipate this SMAD1/5-YAP signaling module to be fundamental in controlling growth and evolution of the amniote cerebral cortex.

SF3B1 Mutations Frequently Occur With Both ATM Mutations and TP53 Mutations In CLL Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2868-2868
Author(s):  
Veronika Navrkalova ◽  
Jitka Malcikova ◽  
Barbara Kantorova ◽  
Ludmila Sebejova ◽  
Karla Plevova ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Hot Spot ◽  
Statistical Significance ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Protein Loss ◽  
Tp53 Mutations ◽  
Active Involvement ◽  
Mutation Profile ◽  
Complete Protein

Abstract Abnormalities of TP53 and ATM genes are well established adverse prognostic markers in CLL. Mutations in splicing factor SF3B1 have recently been described as recurrent and predominantly subclonal aberration. Previous studies inconsistently suggested mutual exclusivity or partial overlap with TP53 mutations. Concerning ATM defects, the association between SF3B1 mutations and del(11q) was reported, but relation to ATM mutations remains unclear. The aims were: (a) to assess association between SF3B1 mutations and the most adverse classic genetic lesions represented by TP53 mutations and del(11q); (b) to investigate association between SF3B1 mutations and ATM mutations in a subset of ATM-characterized patients, and (c) to delineate SF3B1 mutation profile and proportion. We used Sanger sequencing of SF3B1 hot-spot exons 14-16, FASAY analysis of TP53 exons 4-10 and resequencing microarray for ATM mutation detection (all 62 coding exons). We analyzed unfavorable cohort of 338 patients characterized by prevalence of unmutated IGHV (72%). At the time of analysis, 82.5% of the patients were previously untreated. We observed SF3B1 mutation in 17.5% (59/338), TP53 mutation in 20% (68/338), and del(11q) in 27.5% (93/338) of cases. All these genetic defects were significantly more frequent in treated patients (SF3B1: P=0.008, TP53: P<0.0001, del(11q): P=0.0295). Interestingly, we observed quite frequent co-occurrence of SF3B1 and TP53 mutations; 28% (19/68) of p53-affected patients in comparison with 15% (40/270) of p53-wt patients harbored SF3B1 mutation (P=0.019). This co-occurrence was apparent (albeit without statistical significance, P=0.166) also in patients investigated at diagnosis, when 20.6% (7/34) of p53-affected patients but only 11.5% (19/165) of p53-wt patients exhibited SF3B1 mutation. The previously reported increased SF3B1 mutation frequency in patients with del(11q) was also apparent but not significant (P=0.078) in our cohort since mutation frequencies were 24% (22/93) and 15% (37/244) in groups with and without del(11q). To analyze the relation of SF3B1 and ATM mutations we used samples with characterized ATM mutation status (n=112). The p53-defective samples and samples with sole del(11q) were omitted since these more frequently harbored SF3B1 mutation. In the remaining 37 patients we observed that SF3B1 and ATM mutations frequently co-occur: 8/21 ATM-mutated patients (38%) but only 2/16 ATM-TP53-wt patients (12.5%) exhibited SF3B1 mutation (P=0.137). This association should be, however, verified on larger cohort. Concerning the SF3B1 mutation profile, we observed previously reported hot-spot missense mutations with the most abundant mutation K700E (21/59, 36%). In addition, we found 2 short in-frame deletions. Altogether it shows that rather than SF3B1 loss-of-function only partial impairment or gain-of-function is possible. The vast majority of samples had mutation in heterozygous state that correlates with presumably preserved second allele. Interestingly, we found 4 mutations in a proportion around 90% that could be explained by loss of heterozygosity in the locus 2q33.1 by any cause. Our study indicates that SF3B1 mutations frequently overlap with mutations in the DNA damage response pathway genes at least in prognostically unfavorable cohort. It seems that SF3B1 mutations do not lead to complete protein loss indicating rather active involvement of mutated SF3B1 in splicing processes in CLL cells. The work was supported by grants NT13519-4, NT11218-6, MSM0021622430, MUNI/A/0723/2012. Disclosures: No relevant conflicts of interest to declare.

scholarly journals FOXC2 Disease Mutations Identified in Lymphedema Distichiasis Patients Impair Transcriptional Activity and Cell Proliferation

2020 ◽  
Vol 21 (14) ◽  
pp. 5112 ◽  
Author(s):  
Daniela Tavian ◽  
Sara Missaglia ◽  
Sandro Michelini ◽  
Paolo Enrico Maltese ◽  
Elena Manara ◽  
...  
Keyword(s):  
Protein Function ◽  
Clinical Findings ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Autosomal Dominant Disorder ◽  
Mutant Proteins ◽  
Transactivation Activity ◽  
Forkhead Box ◽  
Disease Mutations ◽  
Stop Mutation

FOXC2 is a member of the human forkhead-box gene family and encodes a regulatory transcription factor. Mutations in FOXC2 have been associated with lymphedema distichiasis (LD), an autosomal dominant disorder that primarily affects the limbs. Most patients also show extra eyelashes, a condition known as distichiasis. We previously reported genetic and clinical findings in six unrelated families with LD. Half the patients showed missense mutations, two carried frameshift mutations and a stop mutation was identified in a last patient. Here we analyzed the subcellular localization and transactivation activity of the mutant proteins, showing that all but one (p.Y109*) localized to the nucleus. A significant reduction of transactivation activity was observed in four mutants (p.L80F, p.H199Pfs*264, p.I213Tfs*18, p.Y109*) compared with wild type FOXC2 protein, while only a partial loss of function was associated with p.V228M. The mutant p.I213V showed a very slight increase of transactivation activity. Finally, immunofluorescence analysis revealed that some mutants were sequestered into nuclear aggregates and caused a reduction of cell viability. This study offers new insights into the effect of FOXC2 mutations on protein function and shows the involvement of aberrant aggregation of FOXC2 proteins in cell death.

scholarly journals Alterations in ZnT1 expression and function lead to impaired intracellular zinc homeostasis in cancer

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Adrian Israel Lehvy ◽  
Guy Horev ◽  
Yarden Golan ◽  
Fabian Glaser ◽  
Yael Shammai ◽  
...  
Keyword(s):  
Malignant Tumors ◽  
Zinc Homeostasis ◽  
Healthy Population ◽  
Missense Mutations ◽  
Protein Alignment ◽  
Loss Of Function ◽  
Intracellular Zinc ◽  
Dismal Prognosis ◽  
Zinc Levels ◽  
And Function

Abstract Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.

scholarly journals Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Erin N Asleson ◽  
Dennis M Livingston
Keyword(s):  
Half Life ◽  
Translational Regulation ◽  
Cellular Level ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Proteins ◽  
Gal1 Promoter ◽  
Rad52 Protein ◽  
The Stability ◽  
Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is &gt;2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

scholarly journals Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Seung Won Choi ◽  
Yeri Lee ◽  
Kayoung Shin ◽  
Harim Koo ◽  
Donggeon Kim ◽  
...  
Keyword(s):  
Functional Properties ◽  
Therapeutic Target ◽  
Medical Center ◽  
Dominant Negative ◽  
The Cancer Genome Atlas ◽  
Dominant Negative Effect ◽  
Cell Periphery ◽  
Missense Mutations ◽  
Phosphatase Domain ◽  
Mutant Proteins

AbstractPTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

scholarly journals Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2005
Author(s):  
Irene Vorontsova ◽  
James E. Hall ◽  
Thomas F. Schilling ◽  
Noriaki Nagai ◽  
Yosuke Nakazawa
Keyword(s):  
Cell Adhesion ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Adhesion Assay ◽  
Loss Of Function ◽  
Adhesive Properties ◽  
The Difference ◽  
In Vitro Adhesion ◽  
Cell To Cell Adhesion

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

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