Sequence homology of the Ca2+-dependent regulator of cyclic nucleotide phosphodiesterase from rat testis with other Ca2+-binding proteins.

A Ca2+-dependent regulator protein of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) has previously been isolated from rat testis and shown to be a heat-stable, Ca2+-binding protein with a molecular weight of approximately 17,000. The Ca2+-dependent regulator protein is also structurally similar to troponin-C, the Ca2+-binding component of muscle troponin and Ca2+ mediator of muscle contraction. The present report describes a partial amino acid sequence of the Ca2+-dependent regulator. The protein (148 amino acids) is 50% homologous with skeletal muscle troponin-C, but is 11 residues shorter than the muscle protein. The Ca2+-dependent regulator protein has an NH2-terminal sequence of acetyl-Ala-Asp-Glu, a COOH-terminal sequence of Thr-Ala-Lys and 1 residue of epsilon-trimethyllysine located at position 115. All of these properties are distinct from those of other homologous Ca2+-binding proteins. These properties may account for the biological specificities demonstrated by these proteins as compared to the Ca2+-dependent regulator protein. Based on the sequence and a comparison of the Ca2+-dependent regulator protein to other calcium-binding proteins, our data support the view that all of these moecules contain common sequences, especially at their proposed metal-binding sites.     

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (R_Select) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of R_Select proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.     

Introduction of methyl groups from epsilon-N-methyllysine into the one-carbon pool.

Radioactivity from ingested ?-N-[³H] methyl-and ?-N-[³H]-dimethyllysine residues of reductively methylated casein are rapidly and efficiently absorbed and incorporated into tissues of the chicken. Introduction of label into choline, carnitine and methionine suggests that these amino acids contribute methyl groups to the one-carbon pool.     

Three amino acid substitutions in domain I of calmodulin prevent the activation of chicken smooth muscle myosin light chain kinase.

TaM-BMI is a genetically engineered chimeric protein consisting of the first 55 amino acids of cardiac troponin C (but with the normally inactive first Ca2+ binding domain reactivated by site- directed mutagenesis) ligated to the last three domains of chicken calmodulin (George, S.E., VanBerkum, M.F., Ono, T., Cook, R., Hanley, R.M., Putkey, J.A., and Means, A. R. (1990) J. Biol. Chem. 265, 9228-9235). This protein binds chicken smooth muscle myosin light chain kinase (smMLCK) but fails to activate the enzyme, thus functioning as a potent competitive inhibitor (Ki = 66 nM). We have created 29 mutants of calmodulin designed to identify the minimal number of alterations which must be introduced in the first domain to convert the protein to a competitive inhibitor of smMLCK. Alterations of three amino acids predicted to lie on the external surface of calmodulin (E14A, T34K, S38M) recapitulated the phenotype of TaM-BMI and exhibited a Ki of 38 nM. Both the triple mutant and TaM-BMI activated phosphodiesterase and bound a synthetic peptide analog of the calmodulin binding region of smMLCK with an affinity similar to that of native calmodulin (Kact and Kd values of approximately 2 and 3 nM respectively). When a synthetic peptide analog of the myosin light chain phosphorylation site was used as substrate rather than the 20-kDa light chains, TaM-BMI and the triple mutant were partial agonists: the Km for peptide substrate was increased 100- and 60-fold, and catalytic activity was 45 and 60%, respectively, relative to calmodulin. These data suggest TaM-BMI and E14A/T34K/S38M may interact with the calmodulin binding domain of smMLCK in a manner similar to calmodulin. However, alterations in electrostatic and hydrophobic interactions created by the three amino acid substitutions prevent the conformational change in the enzyme usually produced by calmodulin binding. Lack of such changes results in loss of catalytic activity and light chain binding. Additionally, our results show that altering only 3 amino acids residues converts calmodulin to an enzyme-selective antagonist, thus demonstrating the ability to separate calmodulin binding to smMLCK from calmodulin-induced activation of the enzyme.     

Acetylation of human serum albumin by p-nitrophenyl acetate.

Human serum albumin reacts very rapidly with p-nitrophenyl acetate (NphOAc). Rapid acetylation of the protein accompanies and largely accounts for the easily observed rapid formation of of p-nitrophenolate ion. One group is acetylated much faster than all others. It appears to be located in a high affinity binding site for small fatty acid anions, to have a pKa of 8.7, and a limiting bimolecular rate of reaction with NphOAc of approximately 3 X 10(4) M-1 sec-1 at alkaline pH values. Rapid reversible binding appears to be a major contributor to the high reaction velocity.     

Diverse Host Responses and Outcomes following Simian Immunodeficiency Virus SIVmac239 Infection in Sooty Mangabeys and Rhesus Macaques

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10⁵ to 10⁷ RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8⁺ T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4⁺ T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4⁺ T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.     

Repercussions of the COVID-19 pandemic on athletes: a cross-sectional study

The COVID-19 pandemic has presented significant challenges and implications for the sports community. Thus, this study aimed to describe the prevalence of COVID-19 in Brazilian athletes and identify the epidemiological, clinical, athletic, life and health factors associated with the disease in these individuals. A cross-sectional study was performed involving 414 athletes from 22 different sports using an online questionnaire from August to November 2020. The association between the athletes’ characteristics and COVID-19 was evaluated using a logistic regression model. The prevalence of COVID-19 was 8.5%, although only 40% of athletes reported having been tested. Being under 27 years of age (3-fold), having children (~5-fold), having a teammate test positive for COVID-19 (2.5-fold), and smoking (14-fold) were associated with a possible higher risk of disease. Almost 20% of athletes self-reported musculoskeletal injuries during the period of the pandemic that was studied. Athletes with a university education (P = 0.02), a profession other than sports (P < 0.001), those from a low-income family (P = 0.01), and public health system users (P = 0.04) were significantly less frequently tested for COVID-19, whereas international competitors, athletes who received a wage, and athletes who had a teammate who tested positive for COVID-19 were 2-, 3-, and 15-fold more likely to be tested for COVID-19, respectively. Approximately 26% of the athletes who tested negative or were untested reported more than three characteristic COVID-19 symptoms, and 11% of athletes who tested positive for COVID-19 were asymptomatic. The identification of modifiable (have children, smoking, and teammates positively tested) and non-modifiable (age under 27 years) factors related to COVID-19 in athletes can contribute to implementing surveillance programmes to decrease the incidence of COVID-19 in athletes and its negative impacts in sports.     

MLK3 limits activated Galphaq signaling to Rho by binding to p63RhoGEF

Mixed lineage kinase 3 (MLK3) is a MAP3K that activates the JNK-dependent MAPK pathways. Here, we show that MLK3 is required for cell migration in a manner independent of its role as a MAP3K or MLK3 kinase activity. Rather, MLK3 functions in a regulated way to limit levels of the activated GTPase Rho by binding to the Rho activator, p63RhoGEF/GEFT, which, in turn, prevents its activation by Galphaq. These findings demonstrate a scaffolding role for MLK3 in controlling the extent of Rho activation that modulates cell migration. Moreover, they suggest that MLK3 functions as a network hub that links a number of signaling pathways.