Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T6, T3R(f)3, and R6 As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic β-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.

• MeSH
• beta-buňky * chemie   metabolismus   MeSH
• biologické modely * MeSH
• inzulin * chemie   metabolismus   MeSH
• lidé MeSH
• multimerizace proteinu * MeSH
• neurotransmiterové látky metabolismus   MeSH
• počítačová simulace * MeSH
• sekreční vezikuly * chemie   metabolismus   MeSH
• serotonin metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi-modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping-together artifact of the measurement or an inherent intra-cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra-cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM-algorithm-based method is presented for the conversion of multi-modal to quantal data that provides as by-product estimates of means and variances of basic granule packaging.

• MeSH
• biologie buňky statistika a číselné údaje   MeSH
• cytoplazmatická granula chemie   metabolismus   MeSH
• interpretace statistických dat MeSH
• lidé MeSH
• proteiny metabolismus   MeSH
• sekreční vezikuly chemie   metabolismus   MeSH
• synaptické vezikuly chemie   metabolismus   MeSH
• velikost částic MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca(2+) concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca(2+) sensitivity in endocrine cells, accurate and fast determination of Ca(2+) dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that leads to a robust exocytotic activity. Slow increases in the [Ca(2+)](i) revealed exocytotic phases with different Ca(2+) sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca(2+) sensitivity 'classes' of secretory vesicles have been observed: a highly Ca(2+)-sensitive, a medium Ca(2+)-sensitive and a low Ca(2+)-sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca(2+) sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca(2+) sensor protein.

• MeSH
• beta-buňky fyziologie   chemie   MeSH
• biologické modely MeSH
• chromafinní buňky fyziologie   chemie   MeSH
• cytosol fyziologie   chemie   MeSH
• endokrinní buňky fyziologie   chemie   MeSH
• exocytóza fyziologie   MeSH
• hypofýza cytologie   fyziologie   chemie   MeSH
• intracelulární tekutina fyziologie   chemie   MeSH
• lidé MeSH
• melanotropní buňky cytologie   fyziologie   chemie   MeSH
• metaanalýza jako téma MeSH
• sekreční vezikuly fyziologie   chemie   MeSH
• statistika jako téma MeSH
• vápník fyziologie   chemie   izolace a purifikace   MeSH
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• lidé MeSH
• Publikační typ
• přehledy MeSH