No abstract

No abstract

Background: Shape-function studies are necessary to design better therapeutic alternatives of the plasma gelsolin. Results: N-terminal fragment 30 -161 is the smallest segment with F-actin depolymerization potential, and G1-G3 can function independent of Ca 2ϩ ions or low pH. Conclusion: The g2-g3 linker plays a role in imparting pH/Ca 2ϩ insensitivity to G1-G3. Significance: We provide the first evidence that g2-g3 linker regulates mobility of the G1 domain.

Gelsolin is a key actin cytoskeleton-modulating protein primarily regulated by calcium and phosphoinositides. In addition, low pH has also been suggested to activate gelsolin in the absence of Ca 2؉ ions, although no structural insight on this pathway is available except for a reported decrement in its diffusion coefficient at low pH. We also observed ϳ1.6-fold decrease in the molecular mobility of recombinant gelsolin when buffer pH was lowered from 9 to 5. Analysis of the small angle x-ray scattering data collected over the same pH range indicated that the radius of gyration and maximum linear dimension of gelsolin molecules increased from 30.3 to 34.1 Å and from 100 to 125 Å , respectively. Models generated for each dataset indicated that similar to the Ca 2؉ -induced process, low pH also promotes unwinding of this six-domain protein but only partially. It appeared that pH is able to induce extension of the G1 domain from the rest of the five domains, whereas the Ca 2؉ -sensitive latch between G2 and G6 domains remains closed. Interestingly, increasing the free Ca 2؉ level to merely ϳ40 nM, the partially open pH 5 shape "sprung open" to a shape seen earlier for this protein at pH 8 and 1 mM free Ca 2؉ . Also, pH alone could induce a shape where the g3-g4 linker of gelsolin was open when we truncated the C-tail latch from this protein.Our results provide insight into how under physiological conditions, a drop in pH can fully activate the F-actin-severing shape of gelsolin with micromolar levels of Ca 2؉ available.

HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapR V2 ) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapR V2 to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapR V2 (HapR V2G ), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR V2 and HapR V2G proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a "Y" shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.Studies on the quorum-sensing signal network of Vibrio cholerae have produced a rich harvest of data where the periodic appearance and performance of two regulatory proteins, namely LuxO and HapR, determine the fate of a plethora of disparate cellular events (1). Of these, HapR has been given the status of a master regulator because it controls a wide range of diverse physiological activities, thus exerting a profound influence on the survival and pathogenic potential of this bacterium. Collectively, it represses biofilm development and the production of primary virulence factors (2) while it stimulates the production of HA/protease (3), promotes chitin-induced competence (4), increases resistance to protozoan grazing (5), enhances the survival against oxidative stress (6), and controls the expression of the gene encoding Hcp (7). In a recent effort, Zhu and co-workers have elegantly characterized additional novel direct targets of HapR and illustrated two distinct binding motifs (motif 1 and motif 2) in all target promoters (8). Because it modulates a multitude of diverse cellular parameters, the absence of a functional HapR affects the physiology of V. cholerae to a great extent....

The study aims to map plasma gelsolin (pGSN) levels in diabetic humans and mice models of type II diabetes and to evaluate the efficacy of gelsolin therapy in improvement of diabetes in mice. We report that pGSN values decrease by a factor of 0.45 to 0.5 in the blood of type II diabetic humans and mice models. Oral glucose tolerance test in mice models showed that subcutaneous administration of recombinant pGSN and its F-actin depolymerizing competent versions brought down blood sugar levels comparable to Sitagliptin, a drug used to manage hyperglycemic condition. Further, daily dose of pGSN or its truncated versions to diabetic mice for a week kept sugar levels close to normal values. Also, diabetic mice treated with Sitagliptin for 7 days, showed increase in their pGSN values with the decrease in blood glucose as compared to their levels at the start of treatment. Gelsolin helped in improving glycemic control in diabetic mice. We propose that gelsolin level monitoring and replacement of F-actin severing capable gelsolin(s) should be considered in diabetic care.

No abstract

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes is at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-directed remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize a post-catalytic, high-energy intermediate that drives the organization of the root helix structure within rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.