The crystal and molecular structures of a cathepsin K:chondroitin sulfate complex

Cathepsin K is the major collagenolytic enzyme produced by bone-resorbing osteoclasts. We showed earlier that the unique triple-helical collagen-degrading activity of cathepsin K depends on the formation of complexes with bone-or cartilage-resident glycosaminoglycans, such as chondroitin 4-sulfate (C4-S). Here, we describe the crystal structure of a 1:n complex of cathepsin K:C4-S inhibited by E64 at a resolution of 1.8 Å. The overall structure reveals an unusual “beads-on-a-string”-like organization. Multiple cathepsin K molecules bind specifically to a single cosine curve-shaped strand of C4-S with each cathepsin K molecule interacting with three disaccharide residues of C4-S. One of the more important sets of interactions comes from a single turn of helix close to the N terminus of the proteinase containing a basic amino acid triplet (Arg8-Lys9-Lys10) that forms multiple hydrogen bonds either to the caboxylate or to the 4-sulfate groups of C4-S. Altogether, the binding sites with C4-S are located in the R-domain of cathepsin K and are distant from its active site. This explains why the general proteolytic activity of cathepsin K is not affected by the binding of chondroitin sulfate. Biochemical analyses of cathepsin K and C4-S mixtures support the presence of a 1:n complex in solution; a dissociation constant, K_d, of about 10 nM was determined for the interaction between cathepsin K and C4-S.     

Determinants and trends in perinatal human immunodeficiency virus type 1 (HIV-1) transmission in the metropolitan area of Belo Horizonte, Brazil: 1998 - 2005

Significant decrease in human immunodeficiency virus type 1 (HIV-1) vertical transmission has been observed worldwide in centers where interventions such as antiretroviral therapy (ART), elective cesarean section, and avoidance of breastfeeding have been implemented. This prospective cohort study aimed to assess the determinants of and the temporal trends in HIV-1 vertical transmission in the metropolitan area of Belo Horizonte, Brazil from January 1998 to December 2005. The rate of HIV-1 vertical transmission decreased from 20% in 1998 to 3% in 2005. This decline was associated with increased use of more complex ART regimens during pregnancy. Multivariate analysis restricted to clinical variables demonstrated that non ART, neonatal respiratory distress/sepsis and breastfeeding were independently associated with HIV-1 vertical transmission. When laboratory parameters were included in the model, high maternal viral load and non maternal ART were associated with HIV-1 vertical transmission. The results from this study confirm the impact of ART in the reduction of HIV-1 vertical transmission and indicate the need for improvement in the care and monitoring of mother and infant pairs affected by HIV-1.     

Dietary soy protein inhibits DNA damage and cell survival of colon epithelial cells through attenuated expression of fatty acid synthase

Dietary intake of soy protein decreases tumor incidence in rat models of chemically induced colon cancer. We hypothesized that decreased expression of fatty acid synthase (FASN) underlies, in part, the tumor-preventive effects of soy protein, since FASN overexpression characterizes early tumorigenesis. Here, we show that colonic FASN levels are reduced with dietary intake of soy protein isolate (SPI), compared with a control casein diet, in male Sprague-Dawley rats administered the colon carcinogen azoxymethane. SPI consumption resulted in decreased serum insulin levels and decreased azoxymethane-induced tumor suppressor p53 phosphorylation in colon crypt epithelium. To evaluate potential links between insulin and FASN leading to DNA damage, C2(BBe)1 colon epithelial cells, treated with insulin and/or the carcinogen N-nitroso-N-methylurea (NMU), were evaluated for DNA damage and apoptosis after transfection with control or FASN small interfering RNAs (siRNAs). While the numbers of DNA apurinic/apyrimidinic sites (biomarker of DNA damage) induced by NMU were unaffected by transfection of FASN siRNA, insulin induction of these sites was decreased with FASN knockdown. By contrast, NMU-induced apoptosis of C2(BBe)1, as well as intestinal epithelial cell (IEC)-6, was enhanced by transfected FASN siRNA. Increased FASN expression in IEC-6 cells by addition of liver X receptor agonist T0901317 did not affect apurinic/apyrimidinic site number, but enhanced cell killing by cerulenin, a FASN inhibitor. Moreover, insulin rescued NMU-treated cells from apoptosis in an FASN-dependent manner. Results suggest that dietary SPI, by decreasing circulating insulin levels and colon FASN expression, attenuates insulin-induced DNA damage and FASN-mediated anti-apoptosis during carcinogenesis, resulting in an overall reduced tumorigenic state.     

Regucalcin is expressed in rat mammary gland and prostate and down-regulated by 17β-estradiol

Regucalcin is involved in maintenance of calcium homeostasis due to the activation of Ca²⁺ pumping enzymes in the plasma membrane. It has a suppressive effect in cell proliferation, DNA and RNA synthesis, and may be associated with the abnormal cell division on tumor tissues. On the other hand both estrogens and Ca²⁺ are implicated in breast and prostate cancer but there are no studies focused on the expression of regucalcin in rat mammary gland or prostate. Furthermore, it is known that the expression of regucalcin in rat liver and kidney is regulated by 17β-estradiol (E₂). The aim of this study is to analyze if regucalcin is expressed in rat mammary gland and prostate and if it is regulated by E₂ in these tissues. We demonstrated for the first time that regucalcin mRNA and protein are present in rat mammary gland and prostate by in situ hybridization and immunohistochemistry, respectively. Furthermore, we show by Real-time PCR that E₂ down-regulates regucalcin expression in rat mammary gland and prostate.     

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.     

Dithiocarbazate complexes with the [M(PPh3)] (M═Pd or Pt) moiety: Synthesis, characterization and anti-Tripanosoma cruzi activity

New neutral Pd(II) and Pt(II) complexes of the type [M(L)(PPh₃)] (M═Pd or Pt) were prepared in crystalline form in high-yield synthesis with the S-benzyldithiocarbazates and S-4-nitrobenzyldithiocarbazates derivatives from 2-hydroxyacetophenone, H₂L^1a and H₂L^1b, and benzoylacetone, H₂L^2a and H₂L^2b. The new complexes [Pt(L^1a)(PPh₃)] (1), [Pd(L^1a)(PPh₃)] (2), [Pt(L^1b)(PPh₃)] (3), [Pd(L^1b)(PPh₃)] (4), [Pt(L^2a)(PPh₃)] (5), [Pd(L^2a)(PPh₃)] (6), [Pt(L^2b)(PPh₃)] (7) and [Pd(L^2b)(PPh₃)] (8) were characterized on the basis of elemental analysis, conductivity measurements, UV-visible, IR, electrospray ionization mass spectrometry (ESI-MS), NMR (¹H and ³¹P) and by X-ray diffraction studies. The studies showed that differently from what was observed for the H₂L^1a and H₂L^1b ligands, H₂L^2a and H₂L^2b assume cyclic forms as 5-hydroxypyrazolinic. Upon coordination, H₂L^2a and H₂L^2b suffer ring-opening reaction, coordinating in the same manner as H₂L^1a and H₂L^1b, deprotonated and in O,N,S-tridentate mode to the (MPPh₃)²⁺ moiety. All complexes show a quite similar planar fourfold environment around the M(II) center. Furthermore, these complexes exhibited biological activity on extra and intracellular forms of Trypanosoma cruzi in a time- and concentration-dependent manner with IC₅₀ values ranging from 7.8 to 18.7 μM, while the ligand H₂L^2a presented a trypanocidal activity on trypomastigote form better than the standard drug benznidazole.     

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix α4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.     

Expression and function of αβ1 integrins in pancretic beta (INS-1) cells

Integrin-extracellular matrix interactions are important determinants of beta cell behaviours. The β1 integrin is a well-known regulator of beta cell activities; however, little is known of its associated α subunits. In the present study, αβ1 integrin expression was examined in the rat insulinoma cell line (INS-1) to identify their role in beta cell survival and function. Seven α subunits associated with β1 integrin were identified, including α1-6 and αV. Among these heterodimers, α3β1 was most highly expressed. Common ligands for the α3β1 integrin, including fibronectin, laminin, collagen I and collagen IV were tested to identify the most suitable matrix for INS-1 cell proliferation and function. Cells exposed to collagen I and IV demonstrated significant increases in adhesion, spreading, cell viability, proliferation, and FAK phosphorylation when compared to cells cultured on fibronectin, laminin and controls. Integrin-dependent attachment also had a beneficial effect on beta cell function, increasing Pdx-1 and insulin gene and protein expression on collagens I and IV, in parallel with increased basal insulin release and enhanced insulin secretion upon high glucose challenge. Furthermore, functional blockade of α3β1 integrin decreased cell adhesion, spreading and viability on both collagens and reduced Pdx-1 and insulin expression, indicating that its interactions with collagen matrices are important for beta cell survival and function. These results demonstrate that specific αβ1 integrin-ECM interactions are critical regulators of INS-1 beta cell survival and function and will be important in designing optimal conditions for cell-based therapies for diabetes treatment.     

Biological and functional analysis of statistically significant pathways deregulated in colon cancer by using gene expression profiles

Gene expression profiling offers a great opportunity for studying multi-factor diseases and for understanding the key role of genes in mechanisms which drive a normal cell to a cancer state. Single gene analysis is insufficient to describe the complex perturbations responsible for cancer onset, progression and invasion. A deeper understanding of the mechanisms of tumorigenesis can be reached focusing on deregulation of gene sets or pathways rather than on individual genes. We apply two known and statistically well founded methods for finding pathways and biological processes deregulated in pathological conditions by analyzing gene expression profiles. In particular, we measure the amount of deregulation and assess the statistical significance of predefined pathways belonging to a curated collection (Molecular Signature Database) in a colon cancer data set. We find that pathways strongly involved in different tumors are strictly connected with colon cancer. Moreover, our experimental results show that the study of complex diseases through pathway analysis is able to highlight genes weakly connected to the phenotype which may be difficult to detect by using classical univariate statistics. Our study shows the importance of using gene sets rather than single genes for understanding the main biological processes and pathways involved in colorectal cancer. Our analysis evidences that many of the genes involved in these pathways are strongly associated to colorectal tumorigenesis. In this new perspective, the focus shifts from finding differentially expressed genes to identifying biological processes, cellular functions and pathways perturbed in the phenotypic conditions by analyzing genes co-expressed in a given pathway as a whole, taking into account the possible interactions among them and, more importantly, the correlation of their expression with the phenotypical conditions.