Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome

Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 “cardiac interactome” to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease.     

Characterization of calcium binding properties of lithostathine

The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity. Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation. To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichia coli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy. Equilibrium dialysis with (45)Ca(2+) showed that LIT binds Ca(2+) with 1:1 stoichiometry. EPR studies using the divalent vanadyl (VO(2+)) ion as a paramagnetic substitute for Ca(2+) also showed that VO(2+) binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO(2+) competes with Ca(2+) in binding to LIT. Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95-100%) of binding of Ca(2+) and VO(2+), showing that these residues are critical for calcium binding by LIT.     

Seasonal profile of the diet of the dace Telestes karsticus Marčić & Mrakovčić, 2011 (Cyprinidae,Leuciscinae) endemic to Sušik Creek,Croatia

The first data on the feeding ecology of the endemic Telestes karsticus are presented for specimens sampled monthly (July 2007–July 2008) from the Su?ik Creek in the Lug Polje field, Croatia. Macrozoobenthos density was calculated and compared with the stomach contents of T. karsticus and the seasonal profile of the diet was examined. The ratio of gut length vs. standard length was 0.9 ± 0.12. Gut contents revealed that benthic invertebrates accounted for the majority of food consumed, although terrestrial arthropods, plant material, algae and fish were also found. Since this is the only fish species in the investigated creek, T. karsticus can be concluded to be cannibalistic. The proportion of empty guts found in all seasons was small, indicating year round feeding. The number and diversity of prey species was highest in spring and lowest in summer. Ivlev's electivity index was very high for Cladocera, Coleoptera, Trichoptera, Heteroptera and Hymenoptera in all seasons, suggesting positive selection for these prey groups, as opposed to Oligochaeta, Isopoda and Bivalva, which showed negative values in all seasons. For Actinopterygii, the Ivlev coefficient of selection was highly negative in spring, and highly positive in summer. Plant matter was detected in stomach contents in all seasons, with the highest share found in summer when other prey species were less available. The karstic dace is a generalised feeder, consuming the most available prey, and therefore can be classified as a euryphagous omnivore.     

New vanadium-based magnetic resonance imaging probes: clinical potential for early detection of cancer

We have developed a magnetic resonance imaging (MRI) method for improved detection of cancer with a new class of cancer-specific contrast agents, containing vanadyl (VO²⁺)-chelated organic ligands, specifically bis(acetylacetonato)oxovanadium(IV) [VO(acac)₂]. Vanadyl compounds have been found to accumulate within cells, where they interact with intracellular glycolytic enzymes. Aggressive cancers are metabolically active and highly glycolytic; an MRI contrast agent that enters cells with high glycolytic activity could provide high-resolution functional images of tumor boundaries and internal structure, which cannot be achieved by conventional contrast agents. The present work demonstrates properties of VO(acac)₂ that may give it excellent specificity for cancer detection. A high dose of VO(acac)₂ did not cause any acute or short-term adverse reactions in murine subjects. Calorimetry and spectrofluorometric methods demonstrate that VO(acac)₂ is a blood pool agent that binds to serum albumin with a dissociation constant K _d ~ 2.5 ± 0.7 × 10⁻⁷ M and a binding stoichiometry n = 1.03 ± 0.04. Owing to its prolonged blood half-life and selective leakage from hyperpermeable tumor vasculature, a low dose of VO(acac)₂ (0.15 mmol/kg) selectively enhanced in vivo magnetic resonance images of tumors, providing high-resolution images of their interior structure. The kinetics of uptake and washout are consistent with the hypothesis that VO(acac)₂ preferentially accumulates in cancer cells. Although VO(acac)₂ has a lower relaxivity than gadolinium-based MRI contrast agents, its specificity for highly glycolytic cells may lead to an innovative approach to cancer detection since it has the potential to produce MRI contrast agents that are nontoxic and highly sensitive to cancer metabolism.     

Reproductive biology of the freshwater goby Knipowitschia croatica Mrakovčić, Kerovec,Mišetić & Schneider 1996 (Actinopterygii,Gobiidae)

The reproductive biology and gonadogenesis cycle of the Vrgorac goby, Knipowitschia croatica is described [Conservation of Endangered Freshwater Fish in Europe, Birkhauser Verlag, Basel]. The species displays sexual dimorphism during the spawning period. Sexual maturity is achieved at an early age, with 50% of males and females sexually mature at total lengths of 40–45 mm. Fecundity of gravid females ranged from 188 to 593 eggs, with an egg diameter of 0.22–1.11 mm. Though the extended spawning period lasts from March to November, the highest intensity is observed from April to September. A comparison is made of the reproductive biology of this species with other sand goby species of the genera Knipowitschia and Pomatoschistus.     

GTP-induced conformational changes in translin: a comparison between human and Drosophila proteins

Human translin is a conserved protein, unique in its ability to bind both RNA and DNA. Interestingly, GTP binding has been implicated as a regulator of RNA/DNA binding function of mouse translin (TB-RBP). We cloned and overexpressed the translin orthologue from Drosophila melanogaster and compared its DNA/RNA binding properties in relation to GTP effects with that of human protein. Human translin exhibits a stable octameric state and binds ssDNA/RNA/dsDNA targets, all of which get attenuated when GTP is added. Conversely, Drosophila translin exhibits a stable dimeric state that assembles into a suboctameric (tetramer/hexamer) form and fails to bind ssDNA and RNA targets. Interestingly enough, CD spectral analyses, partial protease digestion profile revealed GTP-specific conformational changes in human translin, whereas the same were largely missing in Drosophila protein. Isothermal calorimetry delineated specific heat changes associated with GTP binding in human translin, which invoked subunit "loosening" in its octamers; the same effect was absent in Drosophila protein. We propose that GTP acts as a specific molecular "switch" that modulates the nucleic acid binding function selectively in human translin, perhaps by affecting its octameric configuration.     

Faster rates of DNA unwinding under alkaline conditions in xrs-5 cells may reflect chromatin structure alterations.

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. The DNA alkaline unwinding technique was used to measure the DNA single-strand breakage caused by gamma-rays in xrs-5 and CHO-K1 cells. Greater rates of DNA unwinding were found in xrs-5 cells as compared to CHO-K1. Independent measurement of DNA strand breakage by DNA filter elution or pulsed-field gel electrophoresis failed to show any difference between the two cell lines. The greater rate of unwinding in xrs-5 cells may reflect an alteration in chromosome structure.     

Structure prediction of a multi-domain EF-hand Ca2+ binding protein by PROPAINOR

PROPAINOR is a new algorithm developed for ab initio prediction of the 3D structures of proteins using knowledge-based nonparametric multivariate statistical methods. This algorithm is found to be most efficient in terms of computational simplicity and prediction accuracy for single-domain proteins as compared to other ab initio methods. In this paper, we have used the algorithm for the atomic structure prediction of a multi-domain (two-domain) calcium-binding protein, whose solution structure has been deposited in the PDB recently (PDB ID: 1JFK). We have studied the sensitivity of the predicted structure to NMR distance restraints with their incorporation as an additional input. Further, we have compared the predicted structures in both these cases with the NMR derived solution structure reported earlier. We have also validated the refined structure for proper stereochemistry and favorable packing environment with good results and elucidated the role of the central linker. Figure The predicted 3D Structure of EhCaBP with bound Ca²⁺ ions (CaBP-0). In the structure, α-helices are shown in pink and the β-strands in yellow. Ca²⁺ ions are depicted as fluorescent green balls. Some of the residues in the calcium-binding loops are depicted in space-fill representation.