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 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 >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 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 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 Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

Abstract 97: Genotype to Phenotype: Function of Common Noncoding and Rare Coding Variants In ANGPTL3

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Xiao Wang ◽  
Avanthi Raghavan ◽  
A. Christina Vourakis ◽  
Alexandra E Sperry ◽  
Wenjun Li ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Association Studies ◽  
Functional Characterization ◽  
Genome Wide Association Studies ◽  
Missense Mutations ◽  
Common Variants ◽  
Loss Of Function ◽  
Wild Type ◽  
Enhancer Activity ◽  
Missense Variants

Human genetics studies have demonstrated a strong link between ANGPTL3 , which encodes lipoprotein lipase inhibitor Angiopoietin-like 3, and blood lipid phenotypes. Rare nonsense ANGPTL3 mutations were identified in patients with familial combined hypolipidemia, while common variants at the ANGPTL3 locus have been found by genome-wide association studies (GWASs) to associate with lower triglycerides (TGs) and low-density lipoprotein cholesterol. In light of the seemingly favorable clinical consequences of ANGPTL3 deficiency, we established an experimental framework to identify (1) causal common variants that regulate ANGPTL3 expression and (2) rare missense mutations that disrupt ANGPTL3 function. Using massively parallel reporter assays, we profiled the regulatory activity of all the common variants linked ( r 2 ≥ 0.5) to the lead GWAS SNP in the ANGPTL3 locus and found that rs10889356 demonstrated significant allele-specific enhancer activity. To validate this finding, we used CRISPR-Cas9 to alter the SNP in a human pluripotent stem cell line. When differentiated into hepatocytes, altered cells displayed a 67% increase in ANGPTL3 expression ( n = 4 wild-type and 4 mutant clones, P = 0.007). CRISPR interference using each of three guide RNAs targeting the SNP in HepG2 cells also substantially increased ANGPTL3 expression. These findings support rs10889356- ANGPTL3 as a causal SNP-gene set. Next, we examined the coding regions of ANGPTL3 in 20,000 sequenced individuals and sought to experimentally define rare missense variants using a mouse model. We used CRISPR-Cas9 to generate Angptl3 knockout mice, which exhibited decreased TG (61%, P < 0.001) and decreased cholesterol (31%, P < 0.002). We reconstituted the knockout mice to normal expression levels with adenoviruses expressing either wild-type ANGPTL3 or missense variant ANGPTL3 . So far we have assessed 28 rare missense variants computationally predicted to be deleterious, of which only 10—D42N, K58E, S117P, P264S, Q286H, L315S, L360Q, T383I, T383S, and Y417C—were validated as loss-of-function (conferring <25% of wild-type activity as assessed by changes in both TG and cholesterol levels), underscoring the need for functional characterization of variants of uncertain significance.

Abnormal WNT5A Signaling Causes Mandibular Hypoplasia in Robinow Syndrome

2017 ◽  
Vol 96 (11) ◽  
pp. 1265-1272 ◽  
Author(s):  
S. Hosseini-Farahabadi ◽  
S.J. Gignac ◽  
A. Danescu ◽  
K. Fu ◽  
J.M. Richman
Keyword(s):  
Cell Migration ◽  
Genetic Diseases ◽  
Adaptor Protein ◽  
In Silico Analysis ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Wild Type ◽  
Mandibular Hypoplasia ◽  
Robinow Syndrome ◽  
The Impact

The study of rare genetic diseases provides valuable insights into human gene function. Here, we investigate dominant Robinow syndrome (RS), which affects the WNT5A signaling pathway. Autosomal dominant RS is caused by missense mutations in WNT5A or nonsense mutations in the adaptor protein DVL1 or DVL3. The recessive form of the disease is caused by loss-of-function mutations in the receptor ROR2. RS is characterized by hypertelorism, midface, and mandibular hypoplasia. Here, we focus on the missense mutations in WNT5A, since the impact on function is difficult to predict from in silico analysis. We used chicken embryo to express wild-type or 2 mutant versions of human WNT5A in the mandible and then examined the morphologic, cellular, and molecular effects. The 3 experimental viruses—wt WNT5A, WNT5AC83S, or WNT5AC182R—all caused shortening of the mandible on the injected side as compared with GFP controls. Although the phenotypes initially appeared similar, we uncovered specific disruption of chondrocyte polarity and shape, inhibition of cell migration, differences in target gene expression, and absence of JNK signaling only in the presence of mutant viruses. In addition, the missense mutations do not appear to block receptor binding, since in paracrine experiments, the mutant protein inhibits cell migration. In this study, we ruled out a straightforward gain or loss of function caused by the WNT5A missense mutations. Instead, the mutations are likely redirecting WNT signaling away from JNK-PCP toward other noncanonical pathways. We conclude that in RS, WNT5A missense mutations have dominant neomorphic effects that interfere with the function of the wild-type protein.

Effect of ARID1a oncogenic loss of function mutations on overall survival in metastatic breast cancer patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13068-e13068
Author(s):  
Veronica Mariotti ◽  
Hatem Hussein Soliman
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Metastatic Breast Cancer ◽  
Cancer Patients ◽  
Metastatic Breast ◽  
Missense Mutations ◽  
Breast Cancer Patients ◽  
Loss Of Function ◽  
Wild Type ◽  
Biologic Effect

e13068 Background: ARID1a (AT Rich Interactive Domain 1A) is part of the SWI/SNF complex, which regulates gene transcription. Its inactivation has been shown to determine resistance to endocrine therapy via enrichment of basal-like gene expression features [Xu G., 2020]. We recently reported that ARID1a lower gene expression might be associated with worse overall survival (OS) [Mariotti V., 2019]. The aim of this study was to analyze the effect of ARID1a mutations on survival in metastatic breast cancer patients (pts). Methods: Breast cancer patients prospectively consented for inclusion in the ORIEN genomic database with known metastatic disease were analyzed using cBioPortal. Predicted biologic effect of mutations from RNASeq data was determined by OncoKB. OS was calculated from initial breast cancer diagnosis to death from any cause. OS analysis using Kaplan Meier and descriptive statistics were performed on SPSS. Results: We identified 644 pts with metastatic breast cancer. Of these, 88 (12.8%) pts harbored an ARID1a mutation. 62 (70.5%) mutations were missense (biologic effect unknown), the remaining 26 (29.5%) were oncogenic loss of function (OLF) changes (frame-shift, deletion, insertion, or nonsense). Median OS was significantly better in patients harboring missense mutations compared to OLF and wild type mutations [median OS 58.2 months (95% CI 44.5-71.8) with missense mutations vs 22.8 months (95% CI 10.8-34.7) with OLF mutations and 27.48 months (95% CI 24.5-30.3) with wild type]. Demographics, tumor features and chemotherapy use were generally equally distributed among the subgroups (table). Conclusions: In our study the median OS was worse in metastatic breast cancer pts harboring OFL ARID1a mutations compared to pts with wild type ARID1a or harboring missense ARID1a mutations. Further studies are warranted to assess how specific ARID1a mutations might affect survival in metastatic breast cancer pts. [Table: see text]

scholarly journals The Role of p53 Signaling in Colorectal Cancer

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2125
Author(s):  
Magdalena C. Liebl ◽  
Thomas G. Hofmann
Keyword(s):  
Colorectal Cancer ◽  
Cancer Progression ◽  
Target Genes ◽  
Tp53 Gene ◽  
Cellular Responses ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Wild Type ◽  
P53 Signaling ◽  
Stress Signals

The transcription factor p53 functions as a critical tumor suppressor by orchestrating a plethora of cellular responses such as DNA repair, cell cycle arrest, cellular senescence, cell death, cell differentiation, and metabolism. In unstressed cells, p53 levels are kept low due to its polyubiquitination by the E3 ubiquitin ligase MDM2. In response to various stress signals, including DNA damage and aberrant growth signals, the interaction between p53 and MDM2 is blocked and p53 becomes stabilized, allowing p53 to regulate a diverse set of cellular responses mainly through the transactivation of its target genes. The outcome of p53 activation is controlled by its dynamics, its interactions with other proteins, and post-translational modifications. Due to its involvement in several tumor-suppressing pathways, p53 function is frequently impaired in human cancers. In colorectal cancer (CRC), the TP53 gene is mutated in 43% of tumors, and the remaining tumors often have compromised p53 functioning because of alterations in the genes encoding proteins involved in p53 regulation, such as ATM (13%) or DNA-PKcs (11%). TP53 mutations in CRC are usually missense mutations that impair wild-type p53 function (loss-of-function) and that even might provide neo-morphic (gain-of-function) activities such as promoting cancer cell stemness, cell proliferation, invasion, and metastasis, thereby promoting cancer progression. Although the first compounds targeting p53 are in clinical trials, a better understanding of wild-type and mutant p53 functions will likely pave the way for novel CRC therapies.

scholarly journals Short Stature is Progressive in Patients with Heterozygous NPR2 Mutations

2020 ◽  
Vol 105 (10) ◽  
pp. 3190-3202 ◽  
Author(s):  
Patrick C Hanley ◽  
Harsh S Kanwar ◽  
Corine Martineau ◽  
Michael A Levine
Keyword(s):  
Short Stature ◽  
Mammalian Cells ◽  
Extended Family ◽  
Medical Center ◽  
Academic Medical Center ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Wild Type ◽  
Z Scores

Abstract Background NPR2 encodes atrial natriuretic peptide receptor B (ANPRB), a regulator of skeletal growth. Biallelic loss-of-function mutations in NPR2 result in acromesomelic dysplasia Maroteaux type (AMDM; OMIM 602875), while heterozygous mutations may account for 2% to 6% of idiopathic short stature (ISS). Objective Describe the physical proportions and growth characteristics of an extended family with novel NPR2 mutations including members with AMDM, ISS, or normal stature. Design and Participants We performed whole exome sequencing in 2 healthy parents and 2 children with AMDM. Detailed genotyping and phenotyping were performed on members of a multigenerational family in an academic medical center. We expressed mutant proteins in mammalian cells and characterized expression and function. Results The sisters with AMDM were compound heterozygotes for missense mutations in the NPR2 gene, a novel p.P93S (maternal) and the previously reported p.R989L (paternal). Both mutant ANPRB proteins were normally expressed in HEK293T cells and exhibited dominant negative effects on wild-type ANPRB catalytic activity. Heterozygous relatives had proportionate short stature (height z-scores −2.06 ± 0.97, median ± SD) compared with their wild-type siblings (−1.37 ± 0.59). Height z-scores progressively and significantly decreased as NPR2-heterozygous children matured, while remaining constant in their wild-type siblings. Conclusions Biallelic NPR2 mutations cause severe skeletal dysplasia (AMDM), whereas heterozygous mutations lead to a subtler phenotype characterized by progressive short stature with by increasing loss of height potential with age.

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