scholarly journals Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism

2019 ◽  
Vol 116 (9) ◽  
pp. 3546-3555 ◽  
Author(s):  
Kimberli J. Kamer ◽  
Wei Jiang ◽  
Virendar K. Kaushik ◽  
Vamsi K. Mootha ◽  
Zenon Grabarek
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Functional Coupling ◽  
Gating Mechanism ◽  
Ef Hand ◽  
Negative Effect ◽  
Oligomeric Complex

The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle’s inner membrane. In mammalian cells the uniporter’s activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1–EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2’s C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2’s function in cells. We propose that in the MICU1–MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.

scholarly journals Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 633-645 ◽  
Author(s):  
Guido Cuperus ◽  
David Shore
Keyword(s):  
Protein A ◽  
Dominant Negative ◽  
Class I ◽  
Dominant Negative Effect ◽  
Rna Pol Ii ◽  
Interacting Protein ◽  
Nucleosome Remodeling ◽  
Negative Effect ◽  
Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals The Diagnostic Journey of a Patient with Prader–Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 875
Author(s):  
Karlijn Pellikaan ◽  
Geeske M. van Woerden ◽  
Lotte Kleinendorst ◽  
Anna G. W. Rosenberg ◽  
Bernhard Horsthemke ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Single Nucleotide Polymorphism Array ◽  
Genetic Defect ◽  
Missense Variant ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Pituitary Hormone ◽  
Negative Effect

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.

Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis

2021 ◽  
pp. 002203452199662
Author(s):  
J.T. Chen ◽  
C.H. Lin ◽  
H.W. Huang ◽  
Y.P. Wang ◽  
P.C. Kao ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Genetic Disorder ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Negative Effect ◽  
Localization Signal ◽  
Gingival Fibromatosis

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

scholarly journals FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

2008 ◽  
Vol 181 (3) ◽  
pp. 497-510 ◽  
Author(s):  
Taichi Hara ◽  
Akito Takamura ◽  
Chieko Kishi ◽  
Shun-ichiro Iemura ◽  
Tohru Natsume ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cellular Functions ◽  
Autophagosome Formation ◽  
Interacting Protein ◽  
Autophagy Induction ◽  
Negative Effect ◽  
And Migration ◽  
Starvation Conditions

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51–like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.

scholarly journals Entamoeba histolyticaExpressing a Dominant Negative N-Truncated Light Subunit of Its Gal-Lectin Are Less Virulent

2002 ◽  
Vol 13 (12) ◽  
pp. 4256-4265 ◽  
Author(s):  
Uriel Katz ◽  
Serge Ankri ◽  
Tamara Stolarsky ◽  
Yael Nuchamowitz ◽  
David Mirelman
Keyword(s):  
Antisense Rna ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Impaired Ability ◽  
Reducing Conditions ◽  
Negative Effect ◽  
Terminal Amino ◽  
Heterodimeric Complex

The 260-kDa heterodimeric Gal/GalNAc-specific Lectin (Gal-lectin) of Entamoeba histolytica dissociates under reducing conditions into a heavy (hgl, 170 kDa) and a light subunit (lgl, 35 kDa). We have previously shown that inhibition of expression of the 35-kDa subunit by antisense RNA causes a decrease in virulence. To further understand the role of the light subunit of the Gal-lectin in pathogenesis, amoebae were transfected with plasmids encoding intact, mutated, and truncated forms of the light subunit lgl1 gene. A transfectant in which the 55 N-terminal amino acids of the lgl were removed, overproduced an N-truncated lgl protein (32 kDa), which replaced most of the native 35-kDa lgl in the formation of the Gal-lectin heterodimeric complex and exerted a dominant negative effect. Amoebae transfected with this construct showed a significant decrease in their ability to adhere to and kill mammalian cells as well as in their capacity to form rosettes with and to phagocytose erythrocytes. In addition, immunofluorescence confocal microscopy of this transfectant with anti–Gal-lectin antibodies showed an impaired ability to cap. These results indicate that the light subunit has a role in enabling the clustering of Gal-lectin complexes and that its N-truncation affects this function, which is required for virulence.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

2008 ◽  
Vol 414 (1) ◽  
pp. 121-131 ◽  
Author(s):  
Richard M. van Rijn ◽  
André van Marle ◽  
Paul L. Chazot ◽  
Ellen Langemeijer ◽  
Yongjun Qin ◽  
...  
Keyword(s):  
Mast Cells ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Inflammatory Diseases ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Effect ◽  
Histamine H4 Receptor ◽  
Negative Effect ◽  
H4 Receptor

The H4R (histamine H4 receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H4R is primarily expressed in eosinophils and mast cells and has the highest homology with the H3R. The occurrence of at least twenty different hH3R (human H3R) isoforms led us to investigate the possible existence of H4R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H4R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H4R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H4R–ligand induced signalling or constitutive activity for these H4R splice variants. However, when co-expressed with full-length H4R [H4R(390) (H4R isoform of 390 amino acids)], the H4R splice variants have a dominant negative effect on the surface expression of H4R(390). We detected H4R(390)–H4R splice varianthetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H4R splice variants were detected in various cell types and expressed at similar levels to the full-length H4R(390) mRNA in, for example, pre-monocytes. We conclude that the H4R splice variants described here have a dominant negative effect on H4R(390) functionality, as they are able to retain H4R(390) intracellularly and inactivate a population of H4R(390), presumably via hetero-oligomerization.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

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