Crystal structure of rat alpha-parvalbumin at 1.05 Angstrom resolution

The crystal structure of rat alpha-parvalbumin has been determined at 1.05 Angstrom resolution, using synchrotron data collected at Advanced Photon Source beamline 19-ID. After refinement with SHELX, employing anisotropic displacement parameters and riding hydrogen atoms, R = 0.132 and R(free) = 0.162. The average coordinate estimated standard deviations are 0.021 Angstrom and 0.038 Angstrom for backbone atoms and side-chain atoms, respectively. Besides providing a more precise view of the alpha-isoform than previously available, these data permit comparison with the 0.91 Angstrom structure determined for pike beta-parvalbumin. Visualization of the anisotropic displacement parameters as thermal ellipsoids yields insight into the atomic motion within the Ca(2+)-binding sites. The asymmetric unit includes three parvalbumin (PV) molecules. Interestingly, the EF site in one displays uncharacteristic flexibility. The ellipsoids for Asp-92 are particularly large and non-spherical, and the shape of the Ca(2+) ellipsoid implies significant vibrational motion perpendicular to the plane defined by the four y and z ligands. The relative dearth of crystal-packing interactions in this site suggests that the heightened flexibility may be the result of diminished intermolecular contacts. The implication is that, by impeding conformational mobility, crystal-packing forces may cause serious overestimation of EF-hand rigidity. The high quality of the data permitted 11 residues to be modeled in alternative side-chain conformations, including the two core residues, Ile-97 and Leu-105. The discrete disorder observed for Ile-97 may have functional ramifications, providing a mechanism for communicating binding status between the CD and EF binding loops and between the PV metal ion-binding domain and the N-terminal AB region.     

High resolution structures of p-aminobenzamidine- and benzamidine-VIIa/soluble tissue factor: unpredicted conformation of the 192-193 peptide bond and mapping of Ca2+, Mg2+, Na+, and Zn2+ sites in factor VIIa

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(2+) ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca(2+) and Mg(2+), and the Ca(2+) sites in FVIIa that could be specifically occupied by Mg(2+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(2+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(2+), Mg(2+), Na(+), and Zn(2+). The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca(2+) and three bound Mg(2+). The EGF1 domain contains one Ca(2+) site, and the protease domain contains one Ca(2+), one Na(+), and two Zn(2+) sites. (45)Ca(2+) binding in the presence/absence of Mg(2+) to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(193) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.     

Estimation of parvalbumin Ca(2+)- and Mg(2+)-binding constants by global least-squares analysis of isothermal titration calorimetry data

The use of competitive isothermal titration calorimetry (ITC) to measure high-affinity binding constants has been largely restricted to systems with a single binding site or multiple identical sites. This study demonstrates the extension of this approach to proteins with two nonequivalent EF-hand Ca(2+)-binding sites--rat beta parvalbumin and the S55D/E59D variant of rat alpha parvalbumin. The method involves simultaneous (global) least-squares analysis of titrations with Ca(2+), with Mg(2+), with Ca(2+) in the presence of Mg(2+), and with Ca(2+) or Mg(2+) in the presence of a competitive chelator (EDTA or EGTA). The Ca(2+) and Mg(2+) binding constants obtained for rat beta agree well with estimates obtained by flow dialysis. Although the Ca(2+) affinity of alpha S55D/E59D is too high to measure by flow dialysis, it was amenable to analysis using the ITC-based approach. The combined S55D and E59D mutations increase the Ca(2+) and Mg(2+) affinities of the mutated binding site by factors of 14 and 26, respectively. This behavior is consistent with that seen previously for the rat beta S55D variant.     

Rat alpha- and beta-parvalbumins: comparison of their pentacarboxylate and site-interconversion variants

Introduction of a fifth carboxylate into the ligand array of the CD site (via the combined S55D and E59D mutations) or the EF site (G98D) of rat alpha-parvalbumin substantially increases divalent ion affinity. This behavior, in conflict with that seen in model peptide systems, agrees with existing data for rat beta-parvalbumin [Henzl et al. (1996) Biochemistry 35, 5856-5869]. The complete analysis of the S55D/E59D double variant necessitated characterization of alpha E59D. Whereas the D59E mutation has minimal influence on beta CD site affinity, E59D has a major impact on the alpha CD site, lowering the apparent association constant by a factor of 14. The thermodynamic consequences of exchanging the rat alpha CD and EF site ligand arrays, which differ at the +z and -x coordination positions, were also examined. When the alpha CD array is imported into the EF site, it acquires a low-affinity phenotype, in agreement with previous findings for beta [Henzl et al. (1998) Biochemistry 37, 9101-9111]. However, when the EF ligand array is introduced into the alpha CD binding loop, it retains a high-affinity signature. This result, contrary to that observed in beta, suggests that the influence of the parvalbumin CD site environment supersedes the intrinsic behavior of the ligand array, a conclusion further supported by the disparate impact of the beta D59E and alpha E59D mutations.     

Genetic polymorphism in the manganese superoxide dismutase gene is associated with an increased risk for hepatocellular carcinoma in HCV-infected Moroccan patients

Hepatocellular carcinoma (HCC) ranks among the 10 most common cancers worldwide. The main risk factors for its development are hepatitis B and C virus infections. Hepatitis B and C viruses induce chronic inflammation and oxidative stress that could predispose a cell to mutagenesis and proliferation. Manganese superoxide dismutase (MnSOD) catalyses the detoxification of free radicals, thus playing a crucial role in the protection against damage. A valine (Val) to alanine (Ala) substitution at amino acid 9, mapping within the mitochondrion-targeting sequence of the MnSOD gene, has been associated with an increased cancer risk. The aim of our study was to investigate a possible association of the Val/Ala-MnSOD polymorphism and HCC development in Moroccan patients. Genotypes were determined by means of PCR and RFLP analysis in 96 patients with HCC and 222 control subjects matched for age, sex, and ethnicity. Homozygous Ala/Ala carriers were 31% in the cases and 18% in the controls, which corresponds to an odds ratio (OR) of 2.89, with a 95% confidence interval (CI) of 1.47-5.68. Stratification into subgroups based on HCV infection status revealed an even more increased risk for homozygous Ala/Ala carriers with hepatitis C infection (38.2% in the cases versus 14.8% in the control subjects OR, 5.09; 95% CI, 1.76-14.66). Our findings provide further evidence of an association between the Ala-9Val MnSOD polymorphism and HCC occurrence in hepatitis C virus-infected Moroccan patients.     

Immune checkpoints in hematologic malignancies: What made the immune cells and clinicians exhausted!

Hematologic malignancies comprise a considerable part of cancers with high mortality at any age. Since the introduction of hematopoietic stem cell transplantation (HSCT), the overall survival of patients dramatically increased. The main goal of HSCT is the induction of a graft-versus-leukemia effect to eradicate the residual cancer cells and also reconstitute a healthy immune system for patients. However, relapse is a nettlesome challenge of HSCT. Like many other tumors, hematologic cancer cells induce immune exhaustion leading to immune escape and relapses after HSCT. Besides malignant cells, inhibitory cells such as tumor-associated macrophages and myeloid-derived suppressor cells express various inhibitory receptors capable of inducing exhaustion in immune cells, especially T and natural killer cells. The significance of immune checkpoint blocking in tumor regression in clinical trials led to the 2018 Nobel Prize in Physiology/Medicine. Here, we reviewed the clinical roles of immune checkpoints in hematologic malignancies and post-HSCT relapses.     

Bilayer Amniotic Membrane/Nano-fibrous Fibroin Scaffold Promotes Differentiation Capability of Menstrual Blood Stem Cells into Keratinocyte-Like Cells

The skin provides a dynamic barrier separating and protecting human body from the exterior world, and then immediate repair and rebuilding of the epidermal barrier is crucial after wound and injury. Wound healing without scars and complete regeneration of skin tissue still remain as a clinical challenge. The demand to engineer scaffolds that actively promote regeneration of damaged areas of the skin has been increased. In this study, menstrual blood-derived stem cells (MenSCs) have been induced to differentiate into keratinocytes-like cells in the presence of human foreskin-derived keratinocytes on a bilayer scaffold based on amniotic membrane and silk fibroin. Based on the findings, newly differentiated keratinocytes from MenSCs successfully expressed the keratinocytes specific markers at both mRNA and protein levels judged by real-time PCR and immunostaining techniques, respectively. We could show that the differentiated cells over bilayer composite scaffolds express the keratinocytes specific markers at higher levels when compared with those cultured in conventional 2D culture system. Based on these findings, bilayer amniotic membrane/nano-fibrous fibroin scaffold represents an efficient natural construct with broad applicability to generate keratinocytes from MenSCs for stem cell-based skin wounds healing and regeneration.     

Association of the AB and CD-EF domains from rat alpha- and beta-parvalbumin

Association of the parvalbumin AB and CD-EF domains was examined in Hepes-buffered saline, pH 7.4, employing fragments from rat alpha and beta. All of the interactions require Ca(2+). In saturating Ca(2+), the alpha AB/alpha CD-EF (alpha/alpha) complex displays an association constant of (7.6 +/- 0.4) x 10(7) M(-1). Ca(2+)-binding data for a mixture of the alpha fragments are compatible with an identical two-site model, yielding an average binding constant of (8.5 +/- 0.2) x 10(5) M(-1). The beta/beta interaction is significantly weaker, exhibiting an association constant of (3.0 +/- 0.6) x 10(6) M(-1). The Ca(2+)-binding constants for beta/beta are likewise diminished, at (1.0 +/- 0.1) x 10(5) and (2.3 +/- 0.2) x 10(4) M(-1). The magnitude of the apparent DeltaDeltaG(degree)' for Ca(2+) binding by alpha/alpha and beta/beta, at 3.4 kcal/mol, approaches that measured for the intact proteins (3.6 kcal/mol) and is substantially larger than the 1.5 kcal/mol value previously measured for the isolated CD-EF domains. This result suggests that the AB domain can modulate the Ca(2+) affinities of the CD and EF sites. Interestingly, the heterologous alpha/beta complex displays a larger association constant [(6.6 +/- 0.4) x 10(6) M(-1)] than the homologous beta/beta complex and heightened Ca(2+) affinity [binding constants of (1.3 +/- 0.1) x 10(6) and (8.8 +/- 0.2) x 10(4) M(-1)]. By contrast, beta/alpha associates more weakly than alpha/alpha and exhibits sharply reduced affinity for Ca(2+). Thus, the interaction between the beta AB domain and beta CD-EF domain may act to attenuate Ca(2+) affinity in the intact protein.