RESUMEN
In recent years, increasing numbers of small proteins have moved into the focus of science. Small proteins have been identified and characterized in all three domains of life, but the majority remains functionally uncharacterized, lack secondary structure, and exhibit limited evolutionary conservation. While quite a few have already been described for bacteria and eukaryotic organisms, the amount of known and functionally analyzed archaeal small proteins is still very limited. In this review, we compile the current state of research, show strategies for systematic approaches for global identification of small archaeal proteins, and address selected functionally characterized examples. Besides, we document exemplarily for one archaeon the tool development and optimization to identify small proteins using genome-wide approaches.
Asunto(s)
Archaea/metabolismo , Proteínas Arqueales/metabolismo , Regulación de la Expresión Génica Arqueal/fisiología , Archaea/genética , Proteínas Arqueales/genética , Genoma ArquealRESUMEN
Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1 ) with high affinity (app. KD = 0.76 µm± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1 . Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1 .
Asunto(s)
Proteínas Arqueales/genética , Glutamato-Amoníaco Ligasa/genética , Methanosarcina/enzimología , Aminoácidos/genética , Regulación de la Expresión Génica Arqueal , Sistemas de Lectura Abierta/genética , ARN Mensajero/genéticaRESUMEN
The high-resolution crystal structure of the flavin-dependent monooxygenase (FMO) from the African locust Zonocerus variegatus is presented and the kinetics of structure-based protein variants are discussed. Z. variegatus expresses three flavin-dependent monooxygenase (ZvFMO) isoforms which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. N-Oxygenation of PAs and the accumulation of PA N-oxides within their haemolymph result in two evolutionary advantages for these insects: (i) they circumvent the defence mechanism of their food plants and (ii) they can use PA N-oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. Here, the crystal structure of the Z. variegatus PA N-oxygenase (ZvPNO), the most active ZvFMO isoform, is reported at 1.6â Å resolution together with kinetic studies of a second isoform, ZvFMOa. This is the first available crystal structure of an FMO from class B (of six different FMO subclasses, A-F) within the family of flavin-dependent monooxygenases that originates from a more highly developed organism than yeast. Despite the differences in sequence between family members, their overall structure is very similar. This indicates the need for high conservation of the three-dimensional structure for this type of reaction throughout all kingdoms of life. Nevertheless, this structure provides the closest relative to the human enzyme that is currently available for modelling studies. Of note, the crystal structure of ZvPNO reveals a unique dimeric arrangement as well as small conformational changes within the active site that have not been observed before. A newly observed kink within helix α8 close to the substrate-binding path might indicate a potential mechanism for product release. The data show that even single amino-acid exchanges in the substrate-entry path, rather than the binding site, have a significant impact on the specific enzyme activity of the isoforms.
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Saltamontes/enzimología , Oxigenasas de Función Mixta/química , Alcaloides de Pirrolicidina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Isoformas de Proteínas/químicaRESUMEN
OBJECTIVE: Few interventions have combined life-style and psychosocial approaches in the context of Type 2 diabetes management. The purpose of this study was to determine the effect of a multicomponent behavioral intervention on weight, glycemic control, renal function, and depressive symptoms in a sample of overweight/obese adults with Type 2 diabetes and marked depressive symptoms. METHODS: A sample of 111 adults with Type 2 diabetes were randomly assigned to a 1-year intervention (n = 57) or usual care (n = 54) in a parallel groups design. Primary outcomes included weight, glycosylated hemoglobin, and Beck Depression Inventory II score. Estimated glomerular filtration rate served as a secondary outcome. All measures were assessed at baseline and 6 and 12 months after randomization by assessors blind to randomization. Latent growth modeling was used to examine intervention effects on each outcome. RESULTS: The intervention resulted in decreased weight (mean [M] = 0.322 kg, standard error [SE] = 0.124 kg, p = .010) and glycosylated hemoglobin (M = 0.066%, SE = 0.028%, p = .017), and Beck Depression Inventory II scores (M = 1.009, SE = 0.226, p < .001), and improved estimated glomerular filtration rate (M = 0.742 ml·min·1.73 m, SE = 0.318 ml·min·1.73 m, p = .020) each month during the first 6 months relative to usual care. CONCLUSIONS: Multicomponent behavioral interventions targeting weight loss and depressive symptoms as well as diet and physical activity are efficacious in the management of Type 2 diabetes. TRIAL REGISTRATION: This study is registered at Clinicaltrials.gov ID: NCT01739205.
Asunto(s)
Terapia Conductista/métodos , Depresión/terapia , Diabetes Mellitus Tipo 2/terapia , Dietoterapia/métodos , Terapia por Ejercicio/métodos , Tasa de Filtración Glomerular/fisiología , Hemoglobina Glucada/metabolismo , Evaluación de Resultado en la Atención de Salud , Sobrepeso/terapia , Conducta de Reducción del Riesgo , Adulto , Anciano , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Obesidad/terapia , Aprendizaje Social , Pérdida de Peso/fisiologíaRESUMEN
Although weight is an important intervention target among patients with metabolic syndrome, few trials have recruited low-income minority populations. The Community Health and Risk-reduction for Metabolic Syndrome randomized controlled trial aimed to examine the effects of a lifestyle intervention on weight and metabolic syndrome components among low-income minority adults. We randomized 120 adults with metabolic syndrome to standard medical care (N = 60) or a lifestyle intervention (N = 60). Using an intent-to-treat approach, we found significant intervention effects on weight [B = -0.452; SE = 0.122; 95 % confidence intervals (CI) -0.653 to -0.251) and glucose levels at 6-months (B = -0.522, SE = 0.234, 95 % CI -0.907 to -0.138). These changes were maintained through the 12-month assessment. No significant effects were observed on insulin resistance or other metabolic syndrome components. Our intervention was successful in achieving modest but significant weight loss and reduction in fasting glucose among low-income minority subjects with metabolic syndrome.
Asunto(s)
Síndrome Metabólico/terapia , Evaluación de Resultado en la Atención de Salud , Pobreza , Conducta de Reducción del Riesgo , Pérdida de Peso , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: This study aimed to determine the association between circulating leptin levels and total depressive symptoms as well as depressive symptom dimensions (cognitive and somatic) after controlling for important confounding factors. METHODS: The study sample was comprised of 135 participants with the metabolic syndrome. Depressive symptoms were measured using the Beck Depression Inventory-II. Leptin was measured using a leptin-specific enzyme immunoassay. Inflammation was assessed using C-reactive protein and interleukin-6 levels. RESULTS: Leptin was significantly associated with somatic depressive symptoms (ß = 0.33, P = 0.018), but not total depressive symptoms (ß = 0.27, P = 0.067) or cognitive depressive symptoms (ß = 0.21, P = 0.182), after controlling for age, gender, body mass index, and insulin resistance. Further adjustment for C-reactive protein and interleukin-6 levels did not alter the relationship (ß = 0.32, P = 0.023) between circulating leptin levels and somatic depressive symptoms. CONCLUSIONS: Leptin is independently associated with somatic depressive symptoms in patients with the metabolic syndrome.