Structure and stability of gamma-crystallins. Denaturation and proteolysis behavior

The denaturation behavior of bovine lens gamma-crystallin fractions II, III, and IV and their susceptibility to proteolysis in vitro was compared to determine whether differences in their stability could play a role in cataract formation. Tertiary and secondary structure changes induced by increasing concentrations of urea, guanidine hydrochloride, and sodium dodecyl sulfate and by increasingly alkaline pH were followed by near-UV and far-UV circular dichroism, Trp fluorescence emission, and exposure of sulfhydryl groups. Major differences were found in the denaturation and proteolysis behavior of the three gamma-crystallin fractions. In general, the unfolding of gamma-II and gamma-III crystallins is rather gradual, suggesting the presence of intermediate unfolding states; in contrast, the order-disorder transition of gamma-IV crystallin is abrupt. The gamma-IV crystallin fraction is the most stable in urea and guanidine hydrochloride, but is most susceptible to nonspecific proteolysis and alkaline pH denaturation. Differences in denaturation and proteolysis behavior are attributed to the inherent differences in the tertiary structures of these crystallins.     

Studies on phospholipase activities in Neurospora crassa conidia

Cytosol retinyl ester lipoprotein complex from rat liver was capable of transferring its unesterified retinol component to serum aporetinol-binding protein. In the presence of serum albumin and aporetinol-binding protein, 68% of retinyl ester was hydrolyzed and up to 30% of unesterified retinol was transferred from cytosol retinyl ester lipoprotein complex to serum aporetinol-binding protein in 24 h at 30 °C. The reconstituted retinol-retinol-binding protein complex showed biochemical and biophysical properties similar to native retinol-retinol-binding protein. Both native and reconstituted retinol-retinol-binding proteins had identical uv, CD, and fluorescence spectra as well as binding affinity to prealbumin. Treatment of cytosol retinyl ester lipoprotein with sulfhydryl reagent, with 1 n NaCl, or with diisopropyl fluorophosphate (0.14 mm) abolished the hydrolysis of retinyl ester; however, the activity of retinol transfer from cytosol retinyl ester lipoprotein complex to serum retinol-binding protein was still unaffected. The activity of retinol transfer was proportional to the amount of retinol content in the complex and the amount of aporetinol-binding protein. These experiments suggest that the cytosol retinyl ester lipoprotein complex serves three major functions: (i) as a storage form of retinyl ester and retinol; (ii) as an enzyme for hydrolyzing its own retinyl ester ligand; and (iii) as a medium for transfer of unesterified retinol to serum retinol-binding protein.     

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg²⁺ ions, EcoRV is able to cleave the DNA but not in presence of Ca²⁺, although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg²⁺(A)-EcoRV-DNA complex compared to Ca²⁺(S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca²⁺(S)-EcoRV-DNA compared to Mg²⁺(A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca²⁺(S) bound complex than in Mg²⁺(A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.     

Sex and STEM Occupation Predict Autism-Spectrum Quotient (AQ) Scores in Half a Million People

This study assesses Autism-Spectrum Quotient (AQ) scores in a ‘big data’ sample collected through the UK Channel 4 television website, following the broadcasting of a medical education program. We examine correlations between the AQ and age, sex, occupation, and UK geographic region in 450,394 individuals. We predicted that age and geography would not be correlated with AQ, whilst sex and occupation would have a correlation. Mean AQ for the total sample score was m = 19.83 (SD = 8.71), slightly higher than a previous systematic review of 6,900 individuals in a non-clinical sample (mean of means = 16.94) This likely reflects that this big-data sample includes individuals with autism who in the systematic review score much higher (mean of means = 35.19). As predicted, sex and occupation differences were observed: on average, males (m = 21.55, SD = 8.82) scored higher than females (m = 18.95; SD = 8.52), and individuals working in a STEM career (m = 21.92, SD = 8.92) scored higher than individuals non-STEM careers (m = 18.92, SD = 8.48). Also as predicted, age and geographic region were not meaningfully correlated with AQ. These results support previous findings relating to sex and STEM careers in the largest set of individuals for which AQ scores have been reported and suggest the AQ is a useful self-report measure of autistic traits.     

Milk-Derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) Promote Adipocyte Differentiation and Inhibit Inflammation in 3T3-F442A Cells

Milk derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) have shown promise as anti-hypertensive agents due to their inhibitory effects on angiotensin converting enzyme (ACE). Due to the key inter-related roles of hypertension, chronic inflammation and insulin resistance in the pathogenesis of metabolic syndrome, there is growing interest in investigating established anti-hypertensive agents for their effects on insulin sensitivity and inflammation. In this study, we examined the effects of IPP and VPP on 3T3-F442A murine pre-adipocytes, a widely used model for studying metabolic diseases. We found that both IPP and VPP induced beneficial adipogenic differentiation as manifested by intracellular lipid accumulation, upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and secretion of the protective lipid hormone adiponectin by these cells. The observed effects were similar to those induced by insulin, suggesting potential benefits in the presence of insulin resistance. IPP and VPP also inhibited cytokine induced pro-inflammatory changes such as reduction in adipokine levels and activation of the nuclear factor kappa B (NF-κB) pathway. Taken together, our findings suggest that IPP and VPP exert insulin-mimetic adipogenic effects and prevent inflammatory changes in adipocytes, which may offer protection against metabolic disease.