scholarly journals Lrrk regulates the dynamic profile of dendritic Golgi outposts through the golgin Lava lamp

2015 ◽  
Vol 210 (3) ◽  
pp. 471-483 ◽  
Author(s):  
Chin-Hsien Lin ◽  
Hsun Li ◽  
Yi-Nan Lee ◽  
Ying-Ju Cheng ◽  
Ruey-Meei Wu ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dendritic Arbor ◽  
Leucine Rich Repeat ◽  
Loss Of Function ◽  
Negative Effects ◽  
Golgi Membranes ◽  
Prominent Component ◽  
Dynamic Profile

Constructing the dendritic arbor of neurons requires dynamic movements of Golgi outposts (GOPs), the prominent component in the dendritic secretory pathway. GOPs move toward dendritic ends (anterograde) or cell bodies (retrograde), whereas most of them remain stationary. Here, we show that Leucine-rich repeat kinase (Lrrk), the Drosophila melanogaster homologue of Parkinson’s disease–associated Lrrk2, regulates GOP dynamics in dendrites. Lrrk localized at stationary GOPs in dendrites and suppressed GOP movement. In Lrrk loss-of-function mutants, anterograde movement of GOPs was enhanced, whereas Lrrk overexpression increased the pool size of stationary GOPs. Lrrk interacted with the golgin Lava lamp and inhibited the interaction between Lva and dynein heavy chain, thus disrupting the recruitment of dynein to Golgi membranes. Whereas overexpression of kinase-dead Lrrk caused dominant-negative effects on GOP dynamics, overexpression of the human LRRK2 mutant G2019S with augmented kinase activity promoted retrograde movement. Our study reveals a pathogenic pathway for LRRK2 mutations causing dendrite degeneration.

Abstract 13164: Tm6sf2 is a Regulator of Liver Fat Metabolism in Smooth Er Influencing Vldl Lipidation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Fei Luo ◽  
Helen H Hobbs ◽  
Jonathan C Cohen
Keyword(s):  
Secretory Pathway ◽  
Fat Metabolism ◽  
Liver Fat ◽  
Cell Fractionation ◽  
Loss Of Function ◽  
Nonalcoholic Fatty Liver ◽  
Golgi Membranes ◽  
Exon 1 ◽  
Rough Er ◽  
Reduced Rate

Backgrounds: A variant in Transmembrane 6 Superfamily Member 2 [ TM6SF2 (E167K)] that is associated with a loss of function is strongly associated with both alcoholic and nonalcoholic fatty liver disease. TM6SF2 is a polytopic protein that is expressed predominantly in the liver and small intestines. Immunolocalization studies are consistent with the protein being present in the endoplasmic reticulum (ER) and the Golgi complex. Tm6sf2 -/- mice replicate the phenotype of individuals with TM6SF2 -167K variant: hepatic steatosis accompanied by hypocholesterolemia, and transaminitis. These mice have a reduced rate of secretion of VLDL-TG without any change in the rate of secretion of ApoB. Thus, TM6SF2 is required for normal lipidation of VLDL. To determine where in the secretory pathway TM6SF2 promotes lipid addition to nascent VLDL, we have generated Tm6sf2 -/- rats. Methods: Two lines of Tm6sf2 -/- rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Rats were fasted 4 hours and tissues were collected and frozen at -80°C. Cell fractionation studies were performed to isolate the smooth and rough ER along with trans - and cis -Golgi membranes. Result: Both lines of rats were confirmed by immunoblotting to produce no TM6SF2 in the liver or intestines. The phenotype of the Tm6sf2 -/- rats resembles that of mice and humans. Cell fractionation studies revealed that TM6SF2 mainly localized to the smooth ER. Unlike what was observed previously by immunocytochemistry, no TM6SF2 was found in the Golgi fraction. Analysis of the lipid profile of the Golgi apparatus in the KO rat revealed that the majority triglyceride (TG) and fatty acids (FA) subclasses were decreased by one to two folds compared to WT in terms of TG or FA to ApoB48 ratios. Conclusions: TM6SF2 is localized predominantly in the smooth ER and regulates the lipidation of VLDL.

scholarly journals Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2

2006 ◽  
Vol 290 (4) ◽  
pp. C1209-C1220 ◽  
Author(s):  
Chung-Jiuan Jeng ◽  
Yu-Ting Chen ◽  
Yi-Wen Chen ◽  
Chih-Yung Tang
Keyword(s):  
Dominant Negative ◽  
Episodic Ataxia ◽  
Molar Ratio ◽  
Loss Of Function ◽  
Negative Effects ◽  
Autosomal Dominant Disorder ◽  
Episodic Ataxia Type 2 ◽  
Episodic Ataxia Type ◽  
Suppression Effects

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming α1A-subunits of human P/Q-type Ca2+ channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human α1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of α1A-WT with each α1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba2+ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of α1A-mutants increased, suggesting the presence of an α1A-mutant-specific suppression effect. When we coexpressed α1A-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of α1A-WT and mutant using a splice variant of α1A-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-α1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects.

scholarly journals Requirement of Cyclin E-Cdk2 Inhibition in p16INK4a-Mediated Growth Suppression

1998 ◽  
Vol 18 (9) ◽  
pp. 5284-5290 ◽  
Author(s):  
Hong Jiang ◽  
Hubert S. Chou ◽  
Liang Zhu
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Tumor Cells ◽  
Kinase Activity ◽  
Cyclin E ◽  
Growth Suppression ◽  
Dominant Negative ◽  
Cyclin D ◽  
Loss Of Function

ABSTRACT Loss-of-function mutations of p16 INK4a have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4N158, designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050–2054, 1993). In this study, we determined functional differences between p16 and Cdk4N158. We show that p16 and Cdk4N158 inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27 KIP1 , while Cdk4N158 formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.

scholarly journals Dominant negative effects of SCN5A missense variants

2021 ◽  
Author(s):  
Matthew J O'Neill ◽  
Ayesha Muhammad ◽  
Bian Li ◽  
Yuko Wada ◽  
Lynn Hall ◽  
...  
Keyword(s):  
Protein Product ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Negative Effects ◽  
Population Database ◽  
Peak Current ◽  
Missense Variants ◽  
Cardiac Sodium Channel ◽  
Negative Effect

Introduction: Up to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A. Recent studies have suggested that the SCN5A protein product NaV1.5 can form dimers and exert dominant negative effects. Methods: We identified 35 LoF variants (<10% peak current compared to wild type (WT)) and 15 partial LoF variants (10-50% peak current compared to WT) that we assessed for dominant negative behavior. SCN5A variants were studied in HEK293T cells alone or in heterozygous co-expression with WT SCN5A using automated patch clamp. To assess clinical risk, we compared the prevalence of dominant negative vs. putative haploinsufficient (frameshift/splice site) variants in a BrS case consortium and the gnomAD population database. Results: In heterozygous expression with WT, 32/35 LoF variants and 6/15 partial LoF showed reduction to <75% of WT-alone peak INa, demonstrating a dominant negative effect. Carriers of dominant negative LoF missense variants had an enriched disease burden compared to putative haploinsufficient variant carriers (2.7-fold enrichment in BrS cases, p=0.019). Conclusions: Most SCN5A missense LoF variants exert a dominant negative effect. Cohort analyses reveal that this class of variant confers an especially high burden of BrS.

scholarly journals The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation

2012 ◽  
Vol 446 (1) ◽  
pp. 99-111 ◽  
Author(s):  
Iakov N. Rudenko ◽  
Alice Kaganovich ◽  
David N. Hauser ◽  
Aleksandra Beylina ◽  
Ruth Chia ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Missense Mutations ◽  
Leucine Rich Repeat ◽  
Loss Of Function ◽  
Partial Loss ◽  
G2019s Mutation ◽  
Enzymatic Function ◽  
C Terminus

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2–3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.

scholarly journals Defining relative mutational difficulty to understand cancer formation and prevention

2019 ◽  
Author(s):  
Lin Shan ◽  
Jiao Yu ◽  
Zhengjin He ◽  
Shishuang Chen ◽  
Mingxian Liu ◽  
...  
Keyword(s):  
Human Cancer ◽  
Low Frequency ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Nucleotide Change ◽  
P53 Mutations ◽  
Loss Of Function ◽  
Functional Importance ◽  
Negative Effects ◽  
Different Types

SummaryMost mutations in human cancer are low-frequency missense mutations, whose functional status remains hard to predict. Here we show that depending on the type of nucleotide change and the surrounding sequences, the tendency to generate each type of nucleotide mutations varies greatly, even by several hundred folds. Therefore, a cancer-promoting mutation may appear only in a small number of cancer cases, if the underlying nucleotide change is too difficult to generate. We propose a method that integrates both the original mutation counts and their relative mutational difficulty. Using this method, we can accurately predict the functionality of hundreds of low-frequency missense mutations in p53, PTEN and INK4A. Many loss-of-function p53 mutations with dominant negative effects were identified, and the functional importance of several regions in p53 structure were highlighted by this analysis. Furthermore, mutational difficulty analysis also points to potential means of cancer prevention. Our study not only established relative mutational difficulties for different types of mutations in human cancer, but also showed that by incorporating such parameter, we can bring new angles to understanding cancer formation and prevention.

scholarly journals Loss of mutant TP53 does not impair the sustained proliferation, survival or metastasis of diverse cancer cells

2020 ◽  
Author(s):  
Zilu Wang ◽  
François Vaillant ◽  
Catherine Chang ◽  
Chris Riffkin ◽  
Elizabeth Lieschke ◽  
...  
Keyword(s):  
Human Cancer ◽  
Hot Spot ◽  
Point Mutations ◽  
Chemotherapeutic Agents ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Negative Effects ◽  
Substantial Impact ◽  
Human Cancer Cell Lines ◽  
Cellular Stresses

AbstractThe tumour suppressor TP53 is the most frequently mutated gene in human cancer and these aberrations confer poor chemotherapeutic responses1-3. Point mutations typically cluster in the DNA binding domain, with certain ‘hot-spot’ residues disproportionally represented1-4 These mutations abrogate binding of the TP53 transcription factor to DNA and thereby prevent upregulation of genes critical for tumour suppression (loss-of-function)1-3. Mutant TP53 is reported to additionally contribute to tumour development, sustained growth and metastasis not only through dominant-negative effects on wild-type TP535 but also through neomorphic gain-of-function (GOF) activities6. Understanding the contributions of these postulated attributes of mutant TP53 to the development and expansion of tumours will facilitate the design of rational therapeutic strategies. Here we used CRISPR/CAS9 to delete mutant TP53 in a panel of diverse human cancer cell lines. The loss of mutant TP53 expression had no impact on the survival, proliferative capacity or metabolic state of the tumour cells, nor did it sensitise them to cellular stresses and chemotherapeutic agents. These data suggest that putative GOF effects of mutant TP53 are not universally required for the sustained survival and proliferation of fully malignant cells. Therefore, therapeutic approaches that abrogate expression or function of mutant TP53 would not be expected to have substantial impact.

scholarly journals Genetic modeling of GNAO1 disorder delineates mechanisms of Gαo dysfunction

2021 ◽  
Author(s):  
Dandan Wang ◽  
Maria Dao ◽  
Brian S Muntean ◽  
Andrew C Giles ◽  
Kirill A Martemyanov ◽  
...  
Keyword(s):  
Neurodevelopmental Disorder ◽  
Molecular Genetic ◽  
Behavioral Model ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Negative Effects ◽  
C Elegans ◽  
Motor Behaviors ◽  
Locomotor Behaviors

Abstract GNAO1 encephalopathy is a neurodevelopmental disorder with a spectrum of symptoms that include dystonic movements, seizures and developmental delay. While numerous GNAO1 mutations are associated with this disorder, the functional consequences of pathological variants are not completely understood. Here, we deployed the invertebrate C. elegans as a whole-animal behavioral model to study the functional effects of GNAO1 disorder-associated mutations. We tested several pathological GNAO1 mutations for effects on locomotor behaviors using a combination of CRISPR/Cas9 gene editing and transgenic overexpression in vivo. We report that all three mutations tested (G42R, G203R and R209C) result in strong loss of function defects when evaluated as homozygous CRISPR alleles. In addition, mutations produced dominant negative effects assessed using both heterozygous CRISPR alleles and transgenic overexpression. Experiments in mice confirmed dominant negative effects of GNAO1 G42R, which impaired numerous motor behaviors. Thus, GNAO1 pathological mutations result in conserved functional outcomes across animal models. Our study further establishes the molecular genetic basis of GNAO1 encephalopathy, and develops a CRISPR-based pipeline for functionally evaluating mutations associated with neurodevelopmental disorders.

scholarly journals Glucocorticoid Receptor β (GRβ): Beyond Its Dominant-Negative Function

2021 ◽  
Vol 22 (7) ◽  
pp. 3649
Author(s):  
Patricia Ramos-Ramírez ◽  
Omar Tliba
Keyword(s):  
Current Knowledge ◽  
Inflammatory Diseases ◽  
Cell Communication ◽  
Cell Types ◽  
The Body ◽  
Dominant Negative ◽  
Negative Effects ◽  
Independent Manner ◽  
Distinct Cell ◽  
Induced Apoptosis

Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRβ, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRβ has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and “directly” regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRβ has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRβ-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRβ and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted.

scholarly journals Hematopoietic Stem Cell Transplantation Resolves the Immune Deficit Associated with STAT3-Dominant-Negative Hyper-IgE Syndrome

2021 ◽  
Author(s):  
Stephanie C. Harrison ◽  
Christo Tsilifis ◽  
Mary A. Slatter ◽  
Zohreh Nademi ◽  
Austen Worth ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Early Report ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Hyper Ige Syndrome

AbstractAutosomal dominant hyper-IgE syndrome caused by dominant-negative loss-of-function mutations in signal transducer and activator of transcription factor 3 (STAT3) (STAT3-HIES) is a rare primary immunodeficiency with multisystem pathology. The quality of life in patients with STAT3-HIES is determined by not only the progressive, life-limiting pulmonary disease, but also significant skin disease including recurrent infections and abscesses requiring surgery. Our early report indicated that hematopoietic stem cell transplantation might not be effective in patients with STAT3-HIES, although a few subsequent reports have reported successful outcomes. We update on progress of our patient now with over 18 years of follow-up and report on an additional seven cases, all of whom have survived despite demonstrating significant disease-related pathology prior to transplant. We conclude that effective cure of the immunological aspects of the disease and stabilization of even severe lung involvement may be achieved by allogeneic hematopoietic stem cell transplantation. Recurrent skin infections and abscesses may be abolished. Donor TH17 cells may produce comparable levels of IL17A to healthy controls. The future challenge will be to determine which patients should best be offered this treatment and at what point in their disease history.

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