The Human Transporter Associated with Antigen Processing: MOLECULAR MODELS TO DESCRIBE PEPTIDE BINDING COMPETENT STATES

The human transporter associated with antigen processing (TAP) is a member of the ATP binding cassette (ABC) transporter superfamily. TAP plays an essential role in the antigen presentation pathway by translocating cytosolic peptides derived from proteasomal degradation into the endoplasmic reticulum lumen. Here, the peptides are loaded into major histocompatibility class I molecules to be in turn exposed at the cell surface for recognition by T-cells. TAP is a heterodimer formed by the association of two half-transporters, TAP1 and TAP2, with a typical ABC transporter core that consists of two nucleotide binding domains and two transmembrane domains. Despite the availability of biological data, a full understanding of the mechanism of action of TAP is limited by the absence of experimental structures of the full-length transporter. Here, we present homology models of TAP built on the crystal structures of P-glycoprotein, ABCB10, and Sav1866. The models represent the transporter in inward- and outward-facing conformations that could represent initial and final states of the transport cycle, respectively. We described conserved regions in the endoplasmic reticulum-facing loops with a role in the opening and closing of the cavity. We also identified conserved π-stacking interactions in the cytosolic part of the transmembrane domains that could explain the experimental data available for TAP1-Phe-265. Electrostatic potential calculations gave structural insights into the role of residues involved in peptide binding, such as TAP1-Val-288, TAP2-Cys-213, TAP2-Met-218. Moreover, these calculations identified additional residues potentially involved in peptide binding, in turn verified with replica exchange simulations performed on a peptide bound to the inward-facing models.     

The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380

Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 A of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2' and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.     

Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.     

Adipocytes IGFBP‐2 Expression in Prepubertal Obese Children

Aims of the study were to measure insulin‐like growth factor‐binding protein‐2 (IGFBP‐2) expression by abdominal subcutaneous adipocytes and to assess the relationship between IGFBP‐2 expression, circulating IGFBP‐2, obesity, and insulin sensitivity in obese children. Thirty‐eight obese children were recruited. Insulin sensitivity was assessed by intravenous glucose tolerance test and body composition by total‐body dual‐energy X‐ray absorptiometry. Serum free and total IGF‐I, IGFBP‐2, adiponectin, and leptin were measured. Relative quantification of IGFBP‐2 mRNA by subcutaneous adipose tissue biopsies was obtained using real‐time PCR. Circulating IGFBP‐2 was positively associated with insulin sensitivity, in agreement with previous studies. IGFBP‐2 expression was associated with fat mass percentage (r = 0.656; P < 0.02), insulin sensitivity (r = ?0.604; P < 0.05), free IGF‐I (r = 0.646; P < 0.05), and leptin (r = 0.603; P < 0.05), but not with circulating IGFBP‐2 (r = 0.003, P = ns). The association between IGFBP‐2 expression and adiposity (r = 0.648; P < 0.05) was independent of insulin sensitivity (covariate). In conclusion, circulating IGFBP‐2 was positively associated with insulin sensitivity. IGFBP‐2 was expressed by subcutaneous abdominal adipocytes of obese children and increased with adiposity, independently from the level of insulin sensitivity. IGFBP‐2 expression may potentially be one of the local mechanisms used by adipocytes to limit further fat gain.     

An interaction between PRRT2 and Na+/K+ ATPase contributes to the control of neuronal excitability

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na⁺/K⁺ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.Subject terms: Proteomics, Cellular neuroscience, Molecular neuroscience, Paediatric neurological disorders     

Structural and ultrastructural evidence for telocytes in prostate stroma

The prostate comprises a glandular epithelium embedded within a fibromuscular stroma. The stroma is a complex arrangement of cells and extracellular matrix (ECM) components in addition to growth factors, regulatory molecules, remodelling enzymes, blood vessels, nerves and immune cells. The principal sources of ECM components are fibroblasts and smooth muscle cells (SMC), which synthesize the structural and regulatory components of the ECM. Telocytes (TCs) were recently described as a novel stromal cell type that exhibited characteristic features. The aim of this study was to confirm the presence of TCs in prostate stromal tissue of gerbils, as the stromal compartment of this gland is a dynamic microenvironment. We used transmission electron microscopy (TEM), light microscopy and immunohistochemistry methods to provide morphological evidence for the presence of TCs. Cells that resembled TCs were observed in gerbil prostatic stroma. These cells had small cellular bodies with very thin and extremely long cellular processes. They were found primarily in the subepithelial area and also at the periphery of SMC layers. TCs also exhibited moniliform processes, caveolae and nuclei surrounded by small amounts of cytoplasm. Close contacts between TC podomers were evident, particularly in the adjacent epithelial compartment. This morphological evidence supported the presence of TCs in the gerbil prostatic stroma, which we report for the first time.     

Human granulocyte colony stimulating factor (hG-CSF): cloning,overexpression, purification and characterization

Background  

Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.     

Prospects and difficulties in the far- and vacuum-ultraviolet spectroscopy of DNA

Typical sources of error in the UV spectroscopy of DNA near and below 200 nm are pointed out. In the case of native DNA a procedure for extending the wavelenght range and for analysing the spectra is proposed. As illustration, spectra for DNAs of various origin recorded in the 164–350 nm range at room temperature and at 110 K are presented.