RESUMO
Adipose tissue, a central player in energy balance, exhibits significant metabolic flexibility that is often compromised in obesity and type 2 diabetes (T2D). Mitochondrial dysfunction within adipocytes leads to inefficient lipid handling and increased oxidative stress, which together promote systemic metabolic disruptions central to obesity and its complications. This review explores the pivotal role that mitochondria play in altering the metabolic functions of the primary adipocyte types, white, brown, and beige, within the context of obesity and T2D. Specifically, in white adipocytes, these dysfunctions contribute to impaired lipid processing and an increased burden of oxidative stress, worsening metabolic disturbances. Conversely, compromised mitochondrial function undermines their thermogenic capabilities, reducing the capacity for optimal energy expenditure in brown adipocytes. Beige adipocytes uniquely combine the functional properties of white and brown adipocytes, maintaining morphological similarities to white adipocytes while possessing the capability to transform into mitochondria-rich, energy-burning cells under appropriate stimuli. Each type of adipocyte displays unique metabolic characteristics, governed by the mitochondrial dynamics specific to each cell type. These distinct mitochondrial metabolic phenotypes are regulated by specialized networks comprising transcription factors, co-activators, and enzymes, which together ensure the precise control of cellular energy processes. Strong evidence has shown impaired adipocyte mitochondrial metabolism and faulty upstream regulators in a causal relationship with obesity-induced T2D. Targeted interventions aimed at improving mitochondrial function in adipocytes offer a promising therapeutic avenue for enhancing systemic macronutrient oxidation, thereby potentially mitigating obesity. Advances in understanding mitochondrial function within adipocytes underscore a pivotal shift in approach to combating obesity and associated comorbidities. Reigniting the burning of calories in adipose tissues, and other important metabolic organs such as the muscle and liver, is crucial given the extensive role of adipose tissue in energy storage and release.
Assuntos
Diabetes Mellitus Tipo 2 , Metabolismo Energético , Mitocôndrias , Obesidade , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Obesidade/metabolismo , Obesidade/patologia , Mitocôndrias/metabolismo , Animais , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Estresse Oxidativo , TermogêneseRESUMO
A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype-phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8â¢chd-7 and NLGN3â¢nlg-1 that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.
Assuntos
Transtorno do Espectro Autista/genética , Moléculas de Adesão Celular Neuronais/genética , Habituação Psicofisiológica/genética , Fenômica/métodos , Animais , Animais Geneticamente Modificados , Transtorno do Espectro Autista/fisiopatologia , Técnicas de Observação do Comportamento/métodos , Comportamento Animal/fisiologia , Caenorhabditis elegans , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Epistasia Genética , Humanos , Imunoglobulinas/genética , Locomoção/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Fenótipo , Fatores de Transcrição/genéticaRESUMO
Establishing metabolic programming begins during fetal and postnatal development, and early-life lipid exposures play a critical role during neonatal adipogenesis. We define how neonatal consumption of a low omega-6 to -3 fatty acid ratio (n6/n3 FA ratio) establishes FA oxidation in adipocyte precursor cells (APCs) before they become adipocytes. In vivo, APCs isolated from mouse pups exposed to the low n6/n3 FA ratio had superior FA oxidation capacity, elevated beige adipocyte mRNAs Ppargc1α, Ucp2, and Runx1, and increased nuclear receptor NR2F2 protein. In vitro, APC treatment with NR2F2 ligand-induced beige adipocyte mRNAs and increased mitochondrial potential but not mass. Single-cell RNA-sequencing analysis revealed low n6/n3 FA ratio yielded more mitochondrial-high APCs and linked APC NR2F2 levels with beige adipocyte signatures and FA oxidation. Establishing beige adipogenesis is of clinical relevance, because fat depots with energetically active, smaller, and more numerous adipocytes improve metabolism and delay metabolic dysfunction.
RESUMO
A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality [like erd-2.1(RNAi); erd-2.2(RNAi)], indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Genes Supressores , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Sítios de Ligação , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Sinapses/metabolismo , Mutações Sintéticas Letais , Proteínas Vesiculares de Transporte de Acetilcolina/química , Proteínas Vesiculares de Transporte de Acetilcolina/genéticaRESUMO
Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na+ and Cl- dependent, has an EC50 value of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/fisiologia , Genes de Helmintos/fisiologia , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/genética , GABAérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA/análise , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Mutação , Ácidos Nipecóticos/farmacologia , Fenótipo , Filogenia , Sódio/metabolismo , Transmissão SinápticaRESUMO
The cho-1 gene in Caenorhabditis elegans encodes a high-affinity plasma-membrane choline transporter believed to be rate limiting for acetylcholine (ACh) synthesis in cholinergic nerve terminals. We found that CHO-1 is expressed in most, but not all cholinergic neurons in C. elegans. cho-1 null mutants are viable and exhibit mild deficits in cholinergic behavior; they are slightly resistant to the acetylcholinesterase inhibitor aldicarb, and they exhibit reduced swimming rates in liquid. cho-1 mutants also fail to sustain swimming behavior; over a 33-min time course, cho-1 mutants slow down or stop swimming, whereas wild-type animals sustain the initial rate of swimming over the duration of the experiment. A functional CHO-1GFP fusion protein rescues these cho-1 mutant phenotypes and is enriched at cholinergic synapses. Although cho-1 mutants clearly exhibit defects in cholinergic behaviors, the loss of cho-1 function has surprisingly mild effects on cholinergic neurotransmission. However, reducing endogenous choline synthesis strongly enhances the phenotype of cho-1 mutants, giving rise to a synthetic uncoordinated phenotype. Our results indicate that both choline transport and de novo synthesis provide choline for ACh synthesis in C. elegans cholinergic neurons.
Assuntos
Acetilcolina/biossíntese , Caenorhabditis elegans/fisiologia , Colina/farmacocinética , Proteínas de Membrana Transportadoras/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Adaptação Fisiológica , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans , Imunofluorescência , Proteínas de Membrana Transportadoras/genética , Neurônios/citologia , Transmissão Sináptica , Distribuição TecidualRESUMO
Human X-ray cross-complementing group 1 (XRCC1) is a single-strand DNA break repair protein which forms a base excision repair (BER) complex with DNA polymerase beta (beta-Pol). Here we report a site- directed mutational analysis in which 16 mutated versions of the XRCC1 N-terminal domain (XRCC1-NTD) were constructed on the basis of previous NMR results that had implicated the proximity of various surface residues to beta-Pol. Mutant proteins defective in XRCC1-NTD interaction with beta-Pol and with a beta-Pol-gapped DNA complex were determined by gel filtration chromatography and a gel mobility shift assay. The interaction surface determined from the mutated residues was found to encompass beta-strand D and E of the five-stranded beta-sheet (betaABGDE) and the protruding alpha2 helix of the XRCC1-NTD. Mutations that included F67A (betaD), E69K (betaD), V86R (betaE) on the five-stranded beta-sheet and deletion of the alpha2 helix, but not mutations within alpha2, abolished binding of the XRCC1-NTD to beta-Pol. A Y136A mutant abolished beta-Pol binding, and a R109S mutant reduced beta-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and delta-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the alpha2 helix and the betaD and betaE strands of XRCC1-NTD to the energetics of beta-Pol binding. Establishing the direct contacts in the beta-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.
Assuntos
DNA Polimerase beta/metabolismo , Proteínas de Ligação a DNA/metabolismo , Substituição de Aminoácidos , Sítios de Ligação/genética , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Solubilidade , Temperatura , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Residues of DNA polymerase beta (beta-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15N/(13)C/(2)H/(1)H,(13)C-methyl(Leu,Ile,Val)-labeled beta-Pol palm-thumb domain was used for assignments of the 1H, 15N, and 13C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A deltaV303-V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of beta-Pol contacts an edge/face region of the beta sheet of the XRCC1 NTD, while the beta-Pol palm weakly contacts the alpha2 helix.
Assuntos
DNA Polimerase beta/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Polimerase beta/química , Primers do DNA , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
The Caenorhabditis elegans unc-2 gene encodes a voltage-gated calcium channel alpha1 subunit structurally related to mammalian dihydropyridine-insensitive high-threshold channels. In the present paper we describe the characterization of seven alleles of unc-2. Using an unc-2 promoter-tagged green fluorescent protein construct, we show that unc-2 is primarily expressed in motor neurons, several subsets of sensory neurons, and the HSN and VC neurons that control egg laying. Examination of behavioral phenotypes, including defecation, thrashing, and sensitivities to aldicarb and nicotine suggests that UNC-2 acts presynaptically to mediate both cholinergic and GABAergic neurotransmission. Sequence analysis of the unc-2 alleles shows that e55, ra605, ra606, ra609, and ra610 all are predicted to prematurely terminate and greatly reduce or eliminate unc-2 function. In contrast, the ra612 and ra614 alleles are missense mutations resulting in the substitution of highly conserved residues in the C terminus and the domain IVS4-IVS5 linker, respectively. Heterologous expression of a rat brain P/Q-type channel containing the ra612 mutation shows that the glycine to arginine substitution affects a variety of channel characteristics, including the voltage dependence of activation, steady-state inactivation, as well as channel kinetics. Overall, our findings suggest that UNC-2 plays a pivotal role in mediating a number of physiological processes in the nematode and also defines a number of critical residues important for calcium channel function in vivo.
Assuntos
Alelos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Aldicarb/farmacologia , Substituição de Aminoácidos/genética , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/biossíntese , Canais de Cálcio Tipo N/biossíntese , Canais de Cálcio Tipo N/genética , Linhagem Celular , Análise Mutacional de DNA , Testes Genéticos , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas de Membrana/biossíntese , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Mutação , Neurônios Aferentes/metabolismo , Nicotina/farmacologia , Técnicas de Patch-Clamp , Fenótipo , Ratos , Relação Estrutura-Atividade , TransfecçãoRESUMO
The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colina O-Acetiltransferase/genética , Genes Inseridos , Splicing de RNA , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Animais , Evolução MolecularRESUMO
The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/VAChT and synaptobrevin.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Alelos , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Genes Supressores , Teste de Complementação Genética/métodos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mutação , Sistema Nervoso/metabolismo , Estrutura Terciária de Proteína , Sinapses/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a â¼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Endocitose , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Genes de Helmintos/genética , Genoma/genética , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Fenótipo , Transporte Proteico , Vesículas Sinápticas/ultraestrutura , Sinaptotagminas/metabolismo , Proteínas de Transporte VesicularRESUMO
Neuroligins are postsynaptic cell adhesion proteins that bind specifically to presynaptic membrane proteins called neurexins. Mutations in human neuroligin genes are associated with autism spectrum disorders in some families. The nematode Caenorhabditis elegans has a single neuroligin gene (nlg-1), and approximately a sixth of C. elegans neurons, including some sensory neurons, interneurons and a subset of cholinergic motor neurons, express a neuroligin transcriptional reporter. Neuroligin-deficient mutants of C. elegans are viable, and they do not appear deficient in any major motor functions. However, neuroligin mutants are defective in a subset of sensory behaviors and sensory processing, and are hypersensitive to oxidative stress and mercury compounds; the behavioral deficits are strikingly similar to traits frequently associated with autism spectrum disorders. Our results suggest a possible link between genetic defects in synapse formation or function, and sensitivity to environmental factors in the development of autism spectrum disorders.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Moléculas de Adesão Celular Neuronais/deficiência , Mercúrio/toxicidade , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Sensação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Caenorhabditis elegans/citologia , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Sinais (Psicologia) , Genes Reporter , Humanos , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , TemperaturaRESUMO
Synaptotagmin 1, encoded by the snt-1 gene in Caenorhabditis elegans, is a major synaptic vesicle protein containing two Ca(2+)-binding (C2) domains. Alternative splicing gives rise to two synaptotagmin 1 isoforms, designated SNT-1A and SNT-1B, which differ in amino acid sequence in the third, fourth, and fifth beta-strands of the second C2 domain (C2B). We report here that expression of either SNT-1 isoform under control of a strong pan-neural promoter fully rescues the snt-1 null phenotype. Furthermore, C-terminal fusions of either isoform with GFP are trafficked properly to synapses and are fully functional, unlike synaptotagmin 1Colon, two colonsGFP fusions in mice. Analysis of isoform expression with genomic GFP reporter constructs revealed that the SNT-1A and-1B isoforms are differentially expressed and localized in the C. elegans nervous system. We also report molecular, behavioral, and immunocytochemical analyses of twenty snt-1 mutations. One of these mutations, md259, specifically disrupts expression of the SNT-1A isoform and has defects in a subset of synaptotagmin 1-mediated behaviors. A second mutation, md220, is an in-frame 9-bp deletion that removes a conserved tri-peptide sequence (VIL) in the second beta-strand of the C2B domain and disrupts the proper intracellular trafficking of synaptotagmin. Site-directed mutagenesis of a functional SNT-1Colon, two colonsGFP fusion protein was used to examine the potential role of the VIL sequence in synaptotagmin trafficking. Although our results suggest the VIL sequence is most likely not a specific targeting motif, the use of SNT-1Colon, two colonsGFP fusions has great potential for investigating synaptotagmin trafficking and localization.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sistema Nervoso Central/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Alelos , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Imunofluorescência , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologia , Proteínas Recombinantes de FusãoRESUMO
The light chain 1 (LC1) polypeptide is a member of the leucine-rich repeat protein family and binds at or near the ATP hydrolytic site within the motor domain of the gamma heavy chain from Chlamydomonas outer arm dynein. It consists of an N-terminal helix, a central barrel formed from six leucine-rich repeats that fold as beta beta alpha units, and a C-terminal helical domain that protrudes from the main axis defined by the leucine-rich repeats. Interaction with the gamma heavy chain is likely mediated through a hydrophobic patch on the larger beta sheet face, and the C-terminal region is predicted to insert into the dynein ATP hydrolytic site. Here we have used 1H-15N heteronuclear relaxation measurements obtained at 500 and 600 MHz to refine and validate the LC1 solution structure. In this refined structure, the C-terminal helix is significantly reoriented by more than 20 degrees as compared to the control and provides a more precise understanding of the potential regulatory role of this domain. We also employed the refined structure to perform a dynamic analysis of LC1 using the 600 MHz data set. These results, which were cross validated using the 500 MHz data set, strongly support identification of the predicted LC1 binding surfaces and provide additional insight into the interaction mechanisms of leucine-rich repeat proteins.
Assuntos
Proteínas de Transporte/química , Chlamydomonas/química , Proteínas de Drosophila , Dineínas/química , Proteínas Motores Moleculares/química , Ressonância Magnética Nuclear Biomolecular/métodos , Termodinâmica , Animais , Anisotropia , Cristalografia por Raios X , Flagelos/enzimologia , Modelos Químicos , Modelos Moleculares , Método de Monte Carlo , Isótopos de Nitrogênio , Conformação Proteica , Estrutura Terciária de Proteína , Soluções , Relação Estrutura-AtividadeRESUMO
DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.