RESUMO
Infantile Neuroaxonal Dystrophy (INAD) is a rare neurodegenerative disease that often cuts short the life span of a child to 10 years. With a typical onset at 6 months of age, INAD is characterized by regression of acquired motor skills, delayed motor coordination and eventual loss of voluntary muscle control. Biallelic mutations in the PLA2G6 gene have been identified as the most frequent cause of INAD. We highlight the salient features of INAD molecular pathology and the progress made in molecular diagnostics. We reiterate that enhanced molecular diagnostic methodologies such as targeted gene panel testing, exome sequencing, and whole genome sequencing can help ascertain a molecular diagnosis. We describe how the defective catalytic activity of the PLA2G6 gene could be potentially overcome by enzyme replacement or gene correction, giving examples and challenges specific to INAD. This is expected to encourage steps toward developing and testing emerging therapies that might alleviate INAD progression and help realize objectives of patient formed organizations such as the INADcure Foundation.
RESUMO
Whole genome sequencing has recently revealed the protective effect of a single A2T mutation in heterozygous carriers against Alzheimer's disease (AD) and age-related cognitive decline. The impact of the protective cross-interaction between the wild-type (WT) and A2T variants on the dimer structure is therefore of high interest, as the Aß dimers are the smallest known neurotoxic species. Toward this goal, extensive atomistic replica exchange molecular dynamics simulations of the solvated WT homo- and A2T hetero- Aß1-42 dimers have been performed, resulting into a total of 51 µs of sampling for each system. Weakening of a set of transient, intrachain contacts formed between the central and C-terminal hydrophobic residues is observed in the heterodimeric system. The majority of the heterodimers with reduced interaction between central and C-terminal regions lack any significant secondary structure and display a weak interchain interface. Interestingly, the A2T N-terminus, particularly residue F4, is frequently engaged in tertiary and quaternary interactions with central and C-terminal hydrophobic residues in those distinct structures, leading to hydrophobic burial. This atypical involvement of the N-terminus within A2T heterodimer revealed in our simulations implies possible interference on Aß42 aggregation and toxic oligomer formation, which is consistent with experiments. In conclusion, the present study provides detailed structural insights onto A2T Aß42 heterodimer, which might provide molecular insights onto the AD protective effect of the A2T mutation in the heterozygous state.
Assuntos
Alanina/genética , Substituição de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Treonina/genética , Alanina/química , Doença de Alzheimer/genética , Animais , Humanos , Cinética , Método de Monte Carlo , Mutação/genética , Multimerização Proteica , Estrutura Secundária de Proteína , Treonina/químicaRESUMO
Hepsin, a type II transmembrane serine protease, is upregulated in prostate cancer and known to be involved in the progression of metastasis. Here we report a structure-guided approach, which resulted in the discovery of 2-aryl/pyridin-2-yl-1H-indole derivatives as potent and selective inhibitors of hepsin. Potent and selective inhibition of hepsin by compound 8 is likely due to interactions of the amidine group at the S1 site with the cyclohexyl ring from the 2-aryl group projecting towards the S1' site and the tert-hydroxyl group interacting with His57 side-chain as revealed by X-ray crystallography. Compounds 8 and 10, showed Ki of 0.1 µM for hepsin, and exhibited inhibition of invasion and migration of hepsin-overexpressing cell line. Compounds described here could serve as useful tool reagents to investigate the role of hepsin as a potential therapeutic target in cancer.
Assuntos
Antineoplásicos/farmacologia , Cicloexanos/farmacologia , Indóis/farmacologia , Piridinas/farmacologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cicloexanos/síntese química , Humanos , Indóis/síntese química , Camundongos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Invasividade Neoplásica , Piridinas/síntese química , Inibidores de Serina Proteinase/síntese químicaRESUMO
BACKGROUND: Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is 'Tulsi' (or 'Tulasi' or 'Thulasi') and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were generated for the whole genome sequenced with the Illumina Hiseq 1000, resulting in an assembled genome of 374 Mb, with a genome coverage of 61 % (612 Mb estimated genome size). We have also studied transcriptomes (RNA-Seq) of two subtypes of O. tenuiflorum, Krishna and Rama Tulsi and report the relative expression of genes in both the varieties. RESULTS: The pathways leading to the production of medicinally-important specialized metabolites have been studied in detail, in relation to similar pathways in Arabidopsis thaliana and other plants. Expression levels of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi were observed to be relatively high, explaining the purple colouration of Krishna Tulsi leaves. The expression of six important genes identified from genome data were validated by performing q-RT-PCR in different tissues of five different species, which shows the high extent of urosolic acid-producing genes in young leaves of the Rama subtype. In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases including cancer was confirmed using mass spectrometry. CONCLUSIONS: The availability of the whole genome of O.tenuiflorum and our sequence analysis suggests that small amino acid changes at the functional sites of genes involved in metabolite synthesis pathways confer special medicinal properties to this herb.
Assuntos
Regulação da Expressão Gênica de Plantas , Genoma de Planta , Ocimum/genética , Índia , Ocimum/metabolismo , Folhas de Planta/metabolismo , Plantas Medicinais/genética , Plantas Medicinais/metabolismoRESUMO
Cholesterol is required for the growth and viability of mammalian cells and is an obligate precursor for steroid hormone synthesis. Using a loss-of-function screen for mutants with defects in intracellular cholesterol trafficking, a Chinese hamster ovary cell mutant with haploinsufficiency of the U17 snoRNA was isolated. U17 is an H/ACA orphan snoRNA, for which a function other than ribosomal processing has not previously been identified. Through expression profiling, we identified hypoxia-upregulated mitochondrial movement regulator (HUMMR) mRNA as a target that is negatively regulated by U17 snoRNA. Upregulation of HUMMR in U17 snoRNA-deficient cells promoted the formation of ER-mitochondrial contacts, decreasing esterification of cholesterol and facilitating cholesterol trafficking to mitochondria. U17 snoRNA and HUMMR regulate mitochondrial synthesis of steroids in vivo and are developmentally regulated in steroidogenic tissues, suggesting that the U17 snoRNA-HUMMR pathway may serve a previously unrecognized, physiological role in gonadal tissue maturation.
Assuntos
Colesterol/metabolismo , Proteínas do Olho/biossíntese , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , RNA Nucleolar Pequeno/metabolismo , Regulação para Cima , Animais , Transporte Biológico Ativo/fisiologia , Células CHO , Colesterol/genética , Cricetinae , Cricetulus , Proteínas do Olho/genética , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Células NIH 3T3 , RNA Nucleolar Pequeno/genéticaRESUMO
Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol-sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1(I1061T), encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1(-/-) mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1(-/-) mouse, this Npc1(tm(I1061T)Dso) model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1(I1061T) protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.
Assuntos
Alelos , Proteínas de Transporte/genética , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Animais , Células Cultivadas , Feminino , Técnicas de Introdução de Genes/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína C1 de Niemann-Pick , PrevalênciaRESUMO
The crystal structure of the region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 was determined at a resolution of 2.5 Å. Severe diffraction anisotropy, rotational pseudosymmetry and twinning complicated the refinement of this structure. A systematic explanation confirming the crystal pathologies and describing how the structure was successfully refined is given in this report.
Assuntos
Glicoproteínas/química , Infecções por Rotavirus/virologia , Rotavirus/química , Toxinas Biológicas/química , Proteínas não Estruturais Virais/química , Anisotropia , Cristalografia por Raios X , Humanos , Modelos MolecularesRESUMO
The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 Å. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which unravelled a case of subtle variation between different oligomeric states unknown at the time of solving the structure, are presented in this paper.
Assuntos
Glicoproteínas/química , Estrutura Quaternária de Proteína , Rotavirus/química , Toxinas Biológicas/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A novel pentameric structure which differs from the previously reported tetrameric form of the diarrhea-inducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP4(95-146)) of strain ST3 (ST3:NSP4(95-146)) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca(2+)-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP4(95-146) mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the ΔN72 and ΔN94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of ΔN94 prepared under different conditions, however, revealed protein concentration-dependent but metal ion- and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. ΔN72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions.