RESUMEN
BACKGROUND: Infertility is a worldwide medical issue in which infection is recognized to play a major role. Pathogens trigger various mechanisms that impact fertility, either directly by affecting the physiological indices of semen or indirectly by disrupting the process of spermatogenesis. In the current work, the effect of in-vitro cultivation of Escherichia coli (E. coli), Candida non-albicans (C. non-albicans), and Trichomonas vaginalis (T. vaginalis) (as the most frequently reported sexually transmitted infections) was assessed on the physiological functions of the spermatozoa and the chemical characteristics of the seminal fluid. METHOD: The semen samples were exposed to cultures of E. coli, C. non-albicans, and T. vaginalis. The study analyzed the changes in motility, agglutination, viability, DNA fragmentation index (DFI%), seminal pH, and biochemical parameters at 1/2, 1, 1.5, 2, 2.5, 3.5 and 4 hours. RESULTS: Incubation of the semen samples with E. coli resulted in a progressive increase in agglutination, pH, and nitrite. The seminal glucose and the sperm motility, on the other hand, were reduced. The sperm vitality and seminal protein remained unaffected. C. non-albicans induced three forms of agglutination (head-to-head, tail-to-tail, and head-to-tail), lowered pH values and decreased the sperm motility, but did not alter the seminal protein, glucose, nitrite, nor the spermatozoa viability at the different tested time intervals. T. vaginalis resulted in increased seminal protein, and reduced glucose, pH, and motility. It also induced minimal agglutination and caused unchanged nitrite and sperm viability. The DFI% was increased in all pathogens with the C. non-albicans showing the highest DNA fragmentation index. CONCLUSION: Urogenital infection with E. coli, C. non-albicans, or T. vaginalis is assumed to affect the quality of semen through DNA fragmentation, agglutination and altered seminal chemical microenvironment.
Asunto(s)
Escherichia coli , Semen , Motilidad Espermática , Trichomonas vaginalis , Trichomonas vaginalis/fisiología , Masculino , Humanos , Semen/microbiología , Motilidad Espermática/efectos de los fármacos , Candida/fisiología , Espermatozoides/microbiología , Fragmentación del ADN , Concentración de Iones de HidrógenoRESUMEN
To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.
Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/química , Escherichia coli/química , Escherichia coli/fisiología , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/química , Conformación Proteica , Ribosomas/químicaRESUMEN
The organization and biophysical properties of the cytosol implicitly govern molecular interactions within cells. However, little is known about mechanisms by which cells regulate cytosolic properties and intracellular diffusion rates. Here, we demonstrate that the intracellular environment of budding yeast undertakes a startling transition upon glucose starvation in which macromolecular mobility is dramatically restricted, reducing the movement of both chromatin in the nucleus and mRNPs in the cytoplasm. This confinement cannot be explained by an ATP decrease or the physiological drop in intracellular pH. Rather, our results suggest that the regulation of diffusional mobility is induced by a reduction in cell volume and subsequent increase in molecular crowding which severely alters the biophysical properties of the intracellular environment. A similar response can be observed in fission yeast and bacteria. This reveals a novel mechanism by which cells globally alter their properties to establish a unique homeostasis during starvation.
Asunto(s)
Núcleo Celular/química , Citoplasma/química , Glucosa/metabolismo , Sustancias Macromoleculares/química , Saccharomycetales/fisiología , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Difusión , Saccharomycetales/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiologíaRESUMEN
To investigate the mechanisms by which ß-subunits influence Nav channel function, we solved the crystal structure of the ß2 extracellular domain at 1.35Å. We combined these data with known bacterial Nav channel structural insights and novel functional studies to determine the interactions of specific residues in ß2 with Nav1.2. We identified a flexible loop formed by (72)Cys and (75)Cys, a unique feature among the four ß-subunit isoforms. Moreover, we found that (55)Cys helps to determine the influence of ß2 on Nav1.2 toxin susceptibility. Further mutagenesis combined with the use of spider toxins reveals that (55)Cys forms a disulfide bond with (910)Cys in the Nav1.2 domain II pore loop, thereby suggesting a 1:1 stoichiometry. Our results also provide clues as to which disulfide bonds are formed between adjacent Nav1.2 (912/918)Cys residues. The concepts emerging from this work will help to form a model reflecting the ß-subunit location in a Nav channel complex.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Cristalografía por Rayos X , Análisis Mutacional de ADN , Humanos , Unión Proteica , Conformación Proteica , Mapeo de Interacción de ProteínasRESUMEN
RadA (also known as 'Sms') is a highly conserved protein, found in almost all eubacteria and plants, with sequence similarity to the RecA strand exchange protein and a role in homologous recombination. We investigate here the biochemical properties of the E. coli RadA protein and several mutant forms. RadA is a DNA-dependent ATPase, a DNA-binding protein and can stimulate the branch migration phase of RecA-mediated strand transfer reactions. RadA cannot mediate synaptic pairing between homologous DNA molecules but can drive branch migration to extend the region of heteroduplex DNA, even without RecA. Unlike other branch migration factors RecG and RuvAB, RadA stimulates branch migration within the context of the RecA filament, in the direction of RecA-mediated strand exchange. We propose that RadA-mediated branch migration aids recombination by allowing the 3' invading strand to be incorporated into heteroduplex DNA and to be extended by DNA polymerases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Recombinación Genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMEN
The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.
Asunto(s)
ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Conformación de Ácido Nucleico , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Cristalografía por Rayos X , Drosophila , Eliminación de Gen , Prueba de Complementación Genética , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.
Asunto(s)
Mapas de Interacción de Proteínas , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Retroalimentación Fisiológica , HumanosRESUMEN
CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
Asunto(s)
Canales de Cloruro/química , Canales de Cloruro/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Axones/enzimología , Proteínas de la Membrana/metabolismo , Sinapsis/enzimología , Proteína ADAM10/deficiencia , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Animales , Axones/química , Encéfalo/embriología , Encéfalo/enzimología , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Proteínas de la Membrana/deficiencia , Ratones , Proteoma/análisis , Especificidad por SustratoRESUMEN
Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Membrana Celular/química , Análisis Mutacional de ADN , Glicoforinas/química , Glicoforinas/genética , Glicoforinas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Puntual , Unión Proteica , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Cooperators who pay a cost to produce publically-available benefits can be exploited by cheaters who do not contribute fairly. How might cooperation persist against cheaters? Two classes of mechanisms are known to promote cooperation: 'partner choice', where a cooperator preferentially interacts with cooperative over cheating partners; and 'partner fidelity feedback', where repeated interactions between individuals ensure that cheaters suffer as their cooperative partners languish (see, for example, Momeni et al., 2013). However when both mechanisms can act, differentiating them has generated controversy. Here, I resolve this controversy by noting that selection can operate on organismal and sub-organismal 'entities' such that partner fidelity feedback at sub-organismal level can appear as partner choice at organismal level. I also show that cooperation between multicellular eukaryotes and mitochondria is promoted by partner fidelity feedback and partner choice between sub-organismal entities, in addition to being promoted by partner fidelity feedback between hosts and symbionts, as was previously known.