The kidney plays a crucial role in the maintenance of the body calcium (Ca 2+ ) balance. Ca 2+ is an essential ion in all organisms and participates in a large variety of structural and functional processes. In mammals, active tubular Ca 2+ reabsorption is restricted to the distal part of the nephron, i.e., the late distal convoluted (DCT2) and the connecting tubules (CNT), where approximately 10-15% of the total Ca 2+ is reabsorbed. This active transcellular transport is hallmarked by the transient receptor potential vanilloid 5 (TRPV5) epithelial Ca 2+ channel, regulated by an array of events, and mediated by hormones, including 1,25-dihydroxyvitamin D 3 , parathyroid hormone, and estrogen. Novel molecular mechanisms have been identified, such as the direct regulatory effects of klotho and tissue kallikrein on the abundance of TRPV5 at the apical membrane. The newly discovered mechanisms could provide potential pharmacological targets in the therapy of renal Ca 2+ wasting. This review discusses the three basic molecular steps of active Ca 2+ reabsorption in the DCT/CNT segments of the nephron, including apical entry, cytoplasmic transport, and basolateral extrusion of Ca 2+ . In addition, an overview of the recently identified mechanisms governing this active Ca 2+ transport through the DCT2/CNT epithelial cells will be presented.

These results suggest a modulating effect for klotho primarily restricted to the epithelial Ca(2+) channels TRPV5 and TRPV6.

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4,8,12,16,20 and 24 subunits, each maintaining a strict 3:1 HSPB2:HSPB3 subunit ratio. These complexes are thermally stable up to 40 °C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or αB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could efficiently associate with HSP20. Taken altogether, this study brings evidence that despite the high sequence homology within the sHSP family, the biochemical properties of the HSPB2/B3 complex are distinctly different from other sHSPs, indicating that the HSPB2/B3 assembly likely possesses other cellular functions than its family members. Various mammalian small heat shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintains a strict 3:1 HSPB2:HSPB3 subunit ratio. Analyzing the thermal stability of the HSPB2/B3 assembly by far-UV circular dichroism spectroscopy revealed subtle structural changes, occurring slightly above 40 °C, and an unfolding curve with an inflection point at approximately 56 °C. Furthermore, HSPB2/B3 exerted poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Finally, coimmunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer does not interact with HSP20, HSP27 or αB-crystallin. However, HSPB2 that ...

Crosslinking of proteins by tissue transglutaminase (tTG) is enhanced in amyloid (Ab) deposits characteristic of Alzheimer's disease and sporadic inclusion body myositis. Small heat shock proteins (sHsps) also occur in amyloid deposits. We here report the substrate characteristics for tTG of six sHsps. Hsp27, Hsp20 and HspB8 are both lysine-and glutaminedonors, aB-crystallin only is a lysine-donor, HspB2 a glutaminedonor, and HspB3 no substrate at all. Close interaction of proteins stimulates crosslinking efficiency as crosslinking between different sHsps only takes place within the same heteromeric complex. We also observed that aB-crystallin, Hsp27 and Hsp20 associate with Ab in vitro, and can be readily crosslinked by tTG.

Several o-(trimethylsilyl)aryl imidazolylsulfonates were synthesized in a simple process and successfully applied in cycloadditions involving benzyne intermediates. The precursor offers an efficient alternative for generating benzynes compared to widely used ortho TMS triflates under similar reaction conditions. With the utilization of this new precursor, the formation of potentially genotoxic trifluoromethanesulfonate side product is eliminated. The applicability of the new benzyne precursor was demonstrated in different types of cycloaddition reactions to prepare heterocyclic molecules.