RESUMO
Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using S. cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways.
Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Relação Estrutura-Atividade , Sumoilação/efeitos dos fármacos , Substituição de Aminoácidos/genética , Análise Mutacional de DNA , Mutagênese/genética , Mutagênicos/toxicidade , Ligação Proteica , Saccharomyces cerevisiae/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Tricho-Rhino-Phalangeal syndrome is a rare autosomal dominant genetic disorder caused by mutations in the TRPS1 gene. This malformation syndrome is characterized by distinctive craniofacial features including sparse scalp hair, bulbous tip of the nose, long flat philtrum, thin upper vermilion border, and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations, and short stature. In this report, we describe two patients with the physical manifestations and genotype of TRPS type I but with learning/intellectual disability not typically described as part of the syndrome. The first patient has a novel heterozygous two-base-pair deletion of nucleotides at 3198-3199 (c.3198-3199delAT) in the TRPS1 gene causing a translational frameshift and subsequent alternate stop codon. The second patient has a 3.08 million base-pair interstitial deletion at 8q23.3 (113,735,487-116,818,578), which includes the TRPS1 gene and CSMD3. Our patients have characteristic craniofacial features, Legg-Perthes syndrome, various skeletal abnormalities including cone shaped epiphyses, anxiety (first patient), and intellectual disability, presenting unusual phenotypes that add to the clinical spectrum of the disease.
Assuntos
Proteínas de Ligação a DNA/genética , Disostoses/genética , Deficiência Intelectual/genética , Doença de Legg-Calve-Perthes/genética , Osteocondrodisplasias/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Disostoses/diagnóstico por imagem , Disostoses/fisiopatologia , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/fisiopatologia , Doença de Legg-Calve-Perthes/diagnóstico por imagem , Doença de Legg-Calve-Perthes/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/fisiopatologia , Proteínas Repressoras , Deleção de Sequência , Adulto JovemRESUMO
African Americans are disproportionately burdened by diabetic kidney disease (DKD). However, little is known about the cellular and molecular mechanisms underlying the onset and progression of DKD in this population. The goal of the current study was to determine the association between specific inflammation markers and kidney injury in diabetic African American men. To this end, we recruited diabetic patients either with (n = 20) or without (n = 87) diagnosed kidney disease along with age-matched nondiabetic controls (n = 81). Urinary albumin-to-creatinine ratios (UACRs) and estimated glomerular filtration rates (eGFR) were used for biochemical assessment of kidney function. We then measured plasma and urinary levels of seven inflammatory markers, including adiponectin, C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), interleukin-6 (IL-6), and intercellular cell adhesion molecule-1 (ICAM-1). Plasma levels of TNF-α, TNFR1, and TNFR2 were significantly higher in diabetics with macroalbuminuria compared to nondiabetic controls and diabetics with normoalbuminuria or microalbuminuria. Likewise, urinary levels of ICAM-1 were higher in diabetics with macroalbuminuria compared to the other groups. Indeed, urinary ICAM-1, plasma TNF-α, and adiponectin had moderate positive correlations with UACR while plasma TNFR1 and TNFR2 levels were strongly correlated with kidney injury, indicated by multiple biomarkers of kidney injury. In contrast, though plasma CRP was elevated in diabetic subjects relative to nondiabetic controls, its levels did not correlate with kidney injury. Together, these data suggest that inflammation, particularly that mediated by the TNF-α/NF-κB signaling axis, may play a role in the pathogenesis of DKD in African American men.
Assuntos
Nefropatias Diabéticas/metabolismo , Taxa de Filtração Glomerular/fisiologia , Inflamação/metabolismo , Rim/metabolismo , Adiponectina/metabolismo , Adulto , Negro ou Afro-Americano , Biomarcadores/metabolismo , Proteína C-Reativa/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Humanos , Inflamação/patologia , Inflamação/fisiopatologia , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Rim/patologia , Rim/fisiopatologia , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-N-acetylglucosaminyltransferase (GPI-GlcNAc transferase). The human enzyme has six known subunits, at least four of which, GPI1, PIG-A, PIG-C, and PIG-H, have functional homologs in the budding yeast Saccharomyces cerevisiae. The uncharacterized yeast gene YDR437w encodes a protein with some sequence similarity to human PIG-P, a fifth subunit of the GPI-GlcNAc transferase. Here we show that Ydr437w is a small but essential subunit of the yeast GPI-GlcNAc transferase, and we designate its gene GPI19. Similar to other mutants in the yeast enzyme, temperature-sensitive gpi19 mutants display cell wall defects and hyperactive Ras phenotypes. The Gpi19 protein associates with the yeast GPI-GlcNAc transferase in vivo, as judged by coimmuneprecipitation with the Gpi2 subunit. Moreover, conditional gpi19 mutants are defective for GPI-GlcNAc transferase activity in vitro. Finally, we present evidence for the topology of Gpi19 within the ER membrane.