Molecular bases of cyclic and specific disulfide interchange between human ERO1alpha protein and protein-disulfide isomerase (PDI)

In the endoplasmic reticulum (ER) of human cells, ERO1α and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1α is essential to avoid ER hyperoxidation. To investigate how ERO1α oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding β-hairpin of ERO1α specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b'-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a'-domain. ERO1α associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1α to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1α with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1α-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1α activity may suggest its physiological role in ER redox and protein homeostasis.     

Urotensin II receptor predicts the clinical outcome of prostate cancer patients and is involved in the regulation of motility of prostate adenocarcinoma cells

Urotensin II (UT-II) is a potent vasoconstrictor peptide and its receptor (UTR) was correlated with human cortico-adrenal carcinoma proliferation. In this study, we have evaluated the correlation between UTR expression and prognosis of human prostate adenocarcinoma and the involvement of this receptor in the regulation of biological properties on both in vivo and in vitro models. UTR mRNA and protein, evaluated by real-time PCR and Western blotting, respectively, were expressed at high levels only in androgen-dependent LNCaP cells. In order to investigate UTR changes occurring in human prostate tumorigenesis, we have also evaluated the expression of UTR in vivo in 195 human prostate tissue samples. UTR was always expressed at low intensity in hyperplastic tissues and at high intensity in well-differentiated carcinomas (Gleason 2-3). Moreover, we have evaluated the effects of an antagonist of UTR, urantide on migration and invasion of LNCaP cells. Urantide induced a dose-dependent decrease of motility and invasion of LNCaP cells whose characteristic ameboid movement seems to be advantageous for their malignancy. These effects were paralleled by down-regulating the autophosphorylation of focal adhesion kinase and the integrin surface expression on LNCaP cells. The effects on cell motility and invasion were likely due to the inhibition of RhoA activity induced by both urantide and shRNA UTR. These data suggest that UTR can be considered a prognostic marker in human prostate adenocarcinoma patients.     

Disruption of CD40/CD40L interaction influences the course of Cryptococcus neoformans infection

CD40 signaling has been implicated in various pathogenic processes such as chronic inflammatory disease, graft-versus-host disease, autoimmune disease and cancer. We previously demonstrated in an in vitro system that the CD40/CD40L pathway mediates late interleukin (IL) 12 production in response to Cryptococcus neoformans. The purpose of this study was to examine the course of C. neoformans infection in the absence of CD40/CD40L costimulation. We compared infection in mice genetically lacking CD40L (CD40L(-/-)) and in the wild-type counterpart. The animals were injected intratracheally with C. neoformans and monitored for clearance of the organism and the development of cellular immune response. CD40L(-/-) mice exhibited an exacerbation of infection, evaluated as scarce inflammatory response in the lung, that mirrored an increase of fungal burden. This correlated with impairment of nitrite production and antimicrobial activity by macrophages against C. neoformans and unrelated microorganisms such as Candida albicans. Moreover, IL-12 production by splenic macrophages was diminished in CD40L(-/-) mice and interferon-gamma production by CD4 and CD8 T cells was decreased. CD4 T cells retained the ability to express a costimulatory molecule, CTLA-4, but showed a decrease in CD28 expression. This latter molecule is implicated in a positive effect on proliferation, cytokine production and survival of T cells. Collectively these data demonstrate that absence of CD40L correlates with (i) reduced antimicrobial activity of natural effector cells; (ii) reduction of the magnitude of T cell response; and (iii) increase of fungal growth in the brain. These findings suggest that disruption of CD40/CD40L may be deleterious for development of an efficient immune response to C. neoformans and may identify potential molecular targets for novel immunotherapeutic approaches     

New water soluble pyrroloquinoline derivatives as new potential anticancer agents

A new class of water soluble 3H-pyrrolo[3,2-f]quinoline derivatives has been synthesized and investigated as potential anticancer drugs. Water solubility profiles have been used to select the most promising derivatives. The novel compound 10, having two (2-diethylamino-ethyl) side chains linked through positions 3N and 9O, presents a suitable water solubility profile, and it was shown to exhibit cell growth inhibitory properties when tested against the in-house panel of cell lines, in particular those obtained from melanoma.     

Small hydroxyethylene-based peptidomimetics inhibiting both HIV-1 and C. albicans aspartic proteases

We have extended a highly flexible method for rapidly assembling aspartic protease inhibitors to produce symmetric and asymmetric monohydroxyethylene peptidomimetics. This method is based on the prior synthesis of the central non-cleavable peptide-bond isostere [NH(2)-P(1)psiP1'-NH(2); psi=hydroxyethylene isostere, HNCH(Bz)CHOHCH(2)CH(Bz)NH], with the possibility of accurately controlling its stereochemistry (S,S,S or S,R,S), and subsequently adding appropriate flanking units, chosen from commercially available amino acids, aromatic carboxylic acids, or phenoxyacetic acid (Poa) derivatives. The method was used to make asymmetric inhibitors of general formula Kyn-Xaa-PhepsiPhe-dmPoa, (Kyn=kynurenic acid, Xaa=Val, Thr or D-thienylglycine, M(r)=716-754) and symmetric inhibitors of formula xPoa-PhepsiPhe-xPoa (xPoa=Poa or dimethyl-, hydroxy-, formyl- or acetyl-Poa, M(r)=553-609), with logP(o/w) values ranging from 4.1 to 7.6. Inhibition of HIV-PR did not depend on the stereochemistry of the hydroxyl group, while it depended markedly on the substituents present on the Poa residues, with dmPoa being preferred over Poa or its more hydrophilic derivatives. Conversely, inhibition of Candida albicans Sap2 was higher for the S,S,S epimers, and Poa or its hydrophilic derivatives were preferred over dmPoa.     

Conformational transitions driven by pyridoxal‐5′‐phosphate uptake in the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii

Serine hydroxymethyltransferase (SHMT) is a pyridoxal‐5′‐phosphate (PLP)‐dependent enzyme belonging to the fold type I superfamily, which catalyzes in vivo the reversible conversion of l ‐serine and tetrahydropteroylglutamate (H₄PteGlu) to glycine and 5,10‐methylenetetrahydropteroylglutamate (5,10‐CH₂‐H₄PteGlu). The SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) had been recently purified and characterized. This enzyme was shown to display catalytic and stability properties typical of psychrophilic enzymes, namely high catalytic activity at low temperature and thermolability. To gain deeper insights into the structure–function relationship of piSHMT, the three‐dimensional structure of its apo form was determined by X‐ray crystallography. Homology modeling techniques were applied to build a model of the piSHMT holo form. Comparison of the two forms unraveled the conformation modifications that take place when the apo enzyme binds its cofactor. Our results show that the apo form is in an “open” conformation and possesses four (or five, in chain A) disordered loops whose electron density is not visible by X‐ray crystallography. These loops contain residues that interact with the PLP cofactor and three of them are localized in the major domain that, along with the small domain, constitutes the single subunit of the SHMT homodimer. Cofactor binding triggers a rearrangement of the small domain that moves toward the large domain and screens the PLP binding site at the solvent side. Comparison to the mesophilic apo SHMT from Salmonella typhimurium suggests that the backbone conformational changes are wider in psychrophilic SHMT. Proteins 2014; 82:2831–2841. © 2014 Wiley Periodicals, Inc.     

Does genetic distance between parental species influence outcomes of hybridization among coral reef butterflyfishes?

Christmas Island is located at the overlap of the Indian and Pacific Ocean marine provinces and is a hot spot for marine hybridization. Here, we evaluate the ecological framework and genetic consequences of hybridization between butterflyfishes Chaetodon guttatissimus and Chaetodon punctatofasciatus. Further, we compare our current findings to those from a previous study of hybridization between Chaetodon trifasciatus and Chaetodon lunulatus. For both species groups, habitat and dietary overlap between parental species facilitate frequent heterospecific encounters. Low abundance of potential mates promotes heterospecific pair formation and the breakdown of assortative mating. Despite similarities in ecological frameworks, the population genetic signatures of hybridization differ between the species groups. Mitochondrial and nuclear data from C. guttatissimus × C. punctatofasciatus (1% divergence at cyt b) show bidirectional maternal contributions and relatively high levels of introgression, both inside and outside the Christmas Island hybrid zone. In contrast, C. trifasciatus × C. lunulatus (5% cyt b divergence) exhibit unidirectional mitochondrial inheritance and almost no introgression. Back‐crossing of hybrid C. guttatissimus × C. punctatofasciatus and parental genotypes may eventually confound species‐specific signals within the hybrid zone. In contrast, hybrids of C. trifasciatus and C. lunulatus may coexist with and remain genetically distinct from the parents. Our results, and comparisons with hybridization studies in other reef fish families, indicate that genetic distance between hybridizing species may be a factor influencing outcomes of hybridization in reef fish, which is consistent with predictions from terrestrially derived hybridization theory.     

Functional brain networks: great expectations,hard times and the big leap forward

Many physical and biological systems can be studied using complex network theory, a new statistical physics understanding of graph theory. The recent application of complex network theory to the study of functional brain networks has generated great enthusiasm as it allows addressing hitherto non-standard issues in the field, such as efficiency of brain functioning or vulnerability to damage. However, in spite of its high degree of generality, the theory was originally designed to describe systems profoundly different from the brain. We discuss some important caveats in the wholesale application of existing tools and concepts to a field they were not originally designed to describe. At the same time, we argue that complex network theory has not yet been taken full advantage of, as many of its important aspects are yet to make their appearance in the neuroscience literature. Finally, we propose that, rather than simply borrowing from an existing theory, functional neural networks can inspire a fundamental reformulation of complex network theory, to account for its exquisitely complex functioning mode.