Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.     

Crystal structures of two potent nonamidine inhibitors bound to factor Xa

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.     

Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription

Human bones, recovered from excavations, are an important biological archive of information. In particular, the analysis of the collagen fraction is useful for paleodietary reconstruction, via light stable isotopes, and for (14)C dating. Generally, collagen extraction procedures do not prevent loss of integrity of proteins. As a consequence, information about the state-of-remains preservation is unavailable. Here we describe a "soft" nondestructive CH(3)COOH-based method to recover collagen from archaeological bones, and also to obtain material for successive isotopic analyses. Our isotopic measurements on the extracts indicate that the CH(3)COOH-based method of extraction may be routinely employed in the context of paleodiet studies. In addition, we propose that biochemical characterization by denaturant electrophoresis and Western blot on CH(3)COOH extracts may be used as a bone collagen quality indicator.     

Global change‐driven effects on dissolved organic matter composition: Implications for food webs of northern lakes

Northern ecosystems are experiencing some of the most dramatic impacts of global change on Earth. Rising temperatures, hydrological intensification, changes in atmospheric acid deposition and associated acidification recovery, and changes in vegetative cover are resulting in fundamental changes in terrestrial–aquatic biogeochemical linkages. The effects of global change are readily observed in alterations in the supply of dissolved organic matter (DOM)—the messenger between terrestrial and lake ecosystems—with potentially profound effects on the structure and function of lakes. Northern terrestrial ecosystems contain substantial stores of organic matter and filter or funnel DOM, affecting the timing and magnitude of DOM delivery to surface waters. This terrestrial DOM is processed in streams, rivers, and lakes, ultimately shifting its composition, stoichiometry, and bioavailability. Here, we explore the potential consequences of these global change‐driven effects for lake food webs at northern latitudes. Notably, we provide evidence that increased allochthonous DOM supply to lakes is overwhelming increased autochthonous DOM supply that potentially results from earlier ice‐out and a longer growing season. Furthermore, we assess the potential implications of this shift for the nutritional quality of autotrophs in terms of their stoichiometry, fatty acid composition, toxin production, and methylmercury concentration, and therefore, contaminant transfer through the food web. We conclude that global change in northern regions leads not only to reduced primary productivity but also to nutritionally poorer lake food webs, with discernible consequences for the trophic web to fish and humans.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Breathing space: deoxygenation of aquatic environments can drive differential ecological impacts across biological invasion stages

The influence of climate change on the ecological impacts of invasive alien species (IAS) remains understudied, with deoxygenation of aquatic environments often-overlooked as a consequence of climate change. Here, we therefore assessed how oxygen saturation affects the ecological impact of a predatory invasive fish, the Ponto-Caspian round goby (Neogobius melanostomus), relative to a co-occurring endangered European native analogue, the bullhead (Cottus gobio) experiencing decline in the presence of the IAS. In individual trials and mesocosms, we assessed the effect of high, medium and low (90%, 60% and 30%) oxygen saturation on: (1) functional responses (FRs) of the IAS and native, i.e. per capita feeding rates; (2) the impact on prey populations exerted; and (3) how combined impacts of both fishes change over invasion stages (Pre-invasion, Arrival, Replacement, Proliferation). Both species showed Type II potentially destabilising FRs, but at low oxygen saturation, the invader had a significantly higher feeding rate than the native. Relative Impact Potential, combining fish per capita effects and population abundances, revealed that low oxygen saturation exacerbates the high relative impact of the invader. The Relative Total Impact Potential (RTIP), modelling both consumer species’ impacts on prey populations in a system, was consistently higher at low oxygen saturation and especially high during invader Proliferation. In the mesocosm experiment, low oxygen lowered RTIP where both species were present, but again the IAS retained high relative impact during Replacement and Proliferation stages at low oxygen. We also found evidence of multiple predator effects, principally antagonism. We highlight the threat posed to native communities by IAS alongside climate-related stressors, but note that solutions may be available to remedy hypoxia and potentially mitigate impacts across invasion stages.

     

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency

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Rational redesign of glucose oxidase for improved catalytic function and stability

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.