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 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.

Factors Influencing the Homomeric and Heteromeric Association of Platelet Integrin Transmembrane Domains.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 212-212
Author(s):  
Bryan W. Berger ◽  
Roman Gorelik ◽  
William F. DeGrado ◽  
Joel S. Bennett
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Protein Domain ◽  
Dominant Negative Effect ◽  
Domain Interactions ◽  
The Face ◽  
Negative Effect ◽  
Tm Domain

Abstract Integrin are α/β heterodimers that mediate an array of cell-cell and cell-matrix interactions including platelet adhesion and aggregation. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations that are regulated by transmembrane (TM) domain interactions: when integrins are inactive, the TM domains of their α and β subunits interact; the domains separate when integrins assume their active conformation. Platelets express five α subunits (α2, αIIb, αv, α5, and α6) and two β subunits (β1 and β3) that combine to form five adhesions receptors. Previously, we observed that the αIIb and β3 TM domains undergo both heteromeric and homomeric interactions and there is overlap of the interfaces that mediates these interactions. Less is known about the other platelet integrins. To study their interactions, we used the TOXCAT assay. In TOXCAT, a chimeric protein consisting of an N-terminal ToxR’ DNA binding domain, a C-terminal maltose-binding protein domain, and an interposed TM domain is expressed in the E. coli inner membrane. TM domain-mediated dimerization of the chimeric protein drives the transcriptional activation of a chloramphenicol acetyl transferase (CAT) reporter gene. To enable TOXCAT to measure heteromeric as well as homomeric interactions, we introduced an R68K mutation into the ToxR’ DNA-binding region, thereby preventing CAT synthesis without affecting protein expression. Thus, when both wild-type and disabled ToxR’ are concurrently expressed from the same plasmid, disabled ToxR’ exerts a dominant-negative effect on CAT synthesis. Using this assay, we found that the interaction of platelet integrin TM domains correlated with the presence of a small residue (sr)-xxx-small residue motif (sr-x3-sr) where x = any residue: α2, αIIb and β1, each of which contains a Gx3G motif, had the strongest tendency to undergo specific homomeric association, whereas α2+β1 and αIIb+β3 had the strongest tendency to form heterodimers. In the TM domains of αv, α5 and β3, one or more of the glycines in sr-x3-sr is replaced by Ser or Ala; as a result, homomeric interactions involving these subunits are substantially weaker. Moreover, mutating each of the small residues in sr-x3-sr to Leu precluded the formation of TM domain oligomers, emphasizing the importance of the sr-x3-sr motif. The dominant-negative TOXCAT assay was also used to screen for inactivating αIIbβ3 and αvβ3 mutations. By introducing random mutations into the β3 TM domain and selecting mutants based on a reduction in CAT synthesis, we identified mutations that enhanced heteromeric αIIbβ3 and αvβ3 association. It is noteworthy that mutations that enhanced the interaction of β3 with αIib were present along the face of the β3 TM helix containing the sr-x3-sr motif. By contrast, mutations enhancing αvβ3 association were distributed throughout the β3 TM helix and didn’t cluster around the sr-x3-sr motif. In summary, we have demonstrated that the TM domains of platelet integrin subunits, in addition to αIIb and β3, undergo specific heteromeric and homomeric interactions, suggesting that TM domain interactions may regulate the function of the integrins containing these subunits. Further, our results indicate that sr-x3-sr motifs play an essential role in the oligomerization of these subunits, suggesting that these motifs play a central role in regulating integrin function.

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 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 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 Dominant negative effect of the loss-of-function γ-secretase mutants on the wild-type enzyme through heterooligomerization

2017 ◽  
Vol 114 (48) ◽  
pp. 12731-12736 ◽  
Author(s):  
Rui Zhou ◽  
Guanghui Yang ◽  
Yigong Shi
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Dominant Negative ◽  
Presenilin 1 ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Negative Effect

γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99-residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces β-amyloid (Aβ) peptides. Pathogenic mutations in PSEN1 and PSEN2, which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer’s disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of the WT allele remains to be elucidated. Here we report that each of the loss-of-function γ-secretase variants that carries a PSEN1 mutation suppresses the protease activity of the WT γ-secretase on Aβ production. Each of these γ-secretase variants forms a stable oligomer with the WT γ-secretase in vitro in the presence of the detergent CHAPSO {3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate}, but not digitonin. Importantly, robust protease activity of γ-secretase is detectable in the presence of CHAPSO, but not digitonin. These experimental observations suggest a dominant negative effect of the γ-secretase, in which the protease activity of WT γ-secretase is suppressed by the loss-of-function γ-secretase variants through hetero-oligomerization. The relevance of this finding to the genesis of Alzheimer’s disease is critically evaluated.

Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2709-2718 ◽  
Author(s):  
P. Miskiewicz ◽  
D. Morrissey ◽  
Y. Lan ◽  
L. Raj ◽  
S. Kessler ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Paired Domain ◽  
Mutant Proteins ◽  
Segment Polarity ◽  
Negative Effect

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.

Dominant Negative Form of PAX5 Is Generated by Multimerization of Its DNA Binding Domain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 25-25
Author(s):  
Norihiko Kawamata ◽  
Mario Pennella ◽  
Jennifer Woo ◽  
Arnold Berk ◽  
H. Phillip Koeffler
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Binding Affinity ◽  
Alpha Helix ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Negative Effect ◽  
Dna Binding Affinity

Abstract Abstract 25 We have previously cloned a number of fusion genes involving PAX5 in acute lymphoblastic leukemia (ALL) (Kawamata N. et al. PNAS, 2008). All of these fusion products exerted a dominant negative effect over the wild-type PAX5. One of these fusion PAX5 proteins, PAX5-C20orf112, was generated by the fusion between the DNA binding domain of PAX5 (PAX5DB) and the C-terminal end of C20orf112. To find the mechanism of the dominant negative effect of the PAX5-C20 fusion, we performed Fluorescence Recovery After Photobleaching (FRAP) assay using PAX5-C20 and PAX5wt constructs connected with Yellow Fluorescence Proteins (YFP). Results showed extremely strong DNA binding affinity of PAX5-C20 compared to PAX5wt. FRAP experiments using deletion mutants of PAX5-C20 showed that both the DNA binding domain and C-terminal alpha-helix region of C20 were indispensable for this strong binding to DNA. Fluorescence Resonance Energy Transfer (FRET) assay, Bi-molecule Fluorescence Complementation (BiFC) assay, and co-immunoprecipitation assay showed that C-terminal end of C20 containing an alpha-helix region encodes a homo-multimerization domain. To confirm that homo-multimerization of PAX5DB increases DNA binding affinity, PAX5DB was fused to the inducible dimerization motif of FKBP (PAX5DB-FK). PAX5DB-FK increased its DNA binding affinity with addition of FKBP ligand inducing homo-dimerization. We also fused PAX5DB to homo-dimerization of MAX (bHLH domain), or tetramerization domain of TP53. FRAP assays showed that homo-dimerization increased its DNA binding activity, and homo-tetramerization further increased its DNA binding and its dominant negative effect over PAX5wt. PAX5-ETV6, also a common fusion protein in ALL, exerts a dominant negative effect over PAX5wt. The ETV6 region of this fusion protein has a multimerization (SAM) domain and the PAX5DB-ETV6SAM mutant protein also showed a dominant negative effect and strong binding to DNA. Importantly, in further studies, co-expression of PAX5-C20 and the YFP-C20-alpha-helix-region diminished the strong DNA binding and the dominant negative activity of the fusion protein. Our data show that multimerization of the DNA binding domain of PAX5 induces strong DNA binding activity, leading to its dominant negative effect over the wild type transcription factor. We believe this represents a new paradigm explaining how a number of fusion genes containing a DB motif from one protein and a multimerization motif from the other partner, can behave in a dominant negative fashion. These observations suggest that peptides/ small molecules inhibiting the multimerization of these oncogenic fusion transcription factors can be promising reagents for treating cancers. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document