Matrix metalloproteinase 19 regulates insulin-like growth factor-mediated proliferation,migration, and adhesion in human keratinocytes through proteolysis of insulin-like growth factor binding protein-3

Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal keratinocytes of healthy human skin. The human keratinocyte cell line HaCaT, which like basal keratinocytes constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like growth factor (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-2 or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal keratinocytes.     

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.     

Structural insights into the catalytic mechanism of cyclophilin A

Cyclophilins constitute a ubiquitous protein family whose functions include protein folding, transport and signaling. They possess both sequence-specific binding and proline cis-trans isomerase activities, as exemplified by the interaction between cyclophilin A (CypA) and the HIV-1 CA protein. Here, we report crystal structures of CypA in complex with HIV-1 CA protein variants that bind preferentially with the substrate proline residue in either the cis or the trans conformation. Cis- and trans-Pro substrates are accommodated within the enzyme active site by rearrangement of their N-terminal residues and with minimal distortions in the path of the main chain. CypA Arg55 guanidinium group probably facilitates catalysis by anchoring the substrate proline oxygen and stabilizing sp3 hybridization of the proline nitrogen in the transition state.     

An acidocalcisomal exopolyphosphatase from Leishmania major with high affinity for short chain polyphosphate

We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX). The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa. LmPPX differs from other exopolyphosphatases previously investigated. Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg(2+) requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity. LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, or p-nitrophenylphosphate. Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization. High levels of short chain (21.4 +/- 3.0 mm) and long chain polyP (55.9 +/- 5.6 mm) were detected in L. major promastigotes. The unique characteristics of LmPPX and L. major polyP metabolism may facilitate the development of novel antileishmanial agents.     

A vacuolar-type H+-pyrophosphatase governs maintenance of functional acidocalcisomes and growth of the insect and mammalian forms of Trypanosoma brucei

Vacuolar proton pyrophosphatases (V-H(+)-PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H(+)-PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H(+)-PPase gene by using double-stranded RNA interference methodology to produce inducible V-H(+)-PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H(+)-PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 x 10(5) cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.     

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.     

Understanding muscle coordination of the human leg with dynamical simulations

Muscles coordinate multijoint motion by generating forces that cause reaction forces throughout the body. Thus, a muscle can redistribute existing segmental energy by accelerating some segments and decelerating others. In the process, a muscle may also produce or absorb energy, in which case its summed energetic effect on the segments is positive or negative, respectively. This Borelli Lecture shows how dynamical simulations derived from musculoskeletal models reveal muscle-induced segmental energy redistribution and muscle co-functions and synergies. Synergy occurs when co-excited muscles distribute segmental energy differently to execute the motor task. In maximum height jumping, high vertical velocity at lift-off occurs desirably at full body extension because biarticular leg muscles redistribute the energy produced by the uniarticular leg muscles. In pedaling, synergistic ankle plantarflexor force generation during leg extension allows the high energy produced by the uniarticular hip and knee extensors to be delivered to the crank. An analogous less-powerful flexor synergy exists during leg flexion. Hamstrings reduce crank deceleration during the leg extension-to-flexion transition by not only producing energy but delivering it to the crank through its contribution to the tangential (accelerating) crank force, though this hamstrings function occurs at the opposite (flexion-extension) transition when pedaling backwards. In walking, the eccentric quadriceps activity in early stance not only decelerates the leg but also accelerates the trunk. In mid-stance, the uni- and biarticular plantarflexors, by having opposite segmental energetic effects, act in synergy to support the whole body, so segmental potential and kinetic energy exchange can occur. To conclude, the extraction of unmeasurable variables from dynamical simulations emulating task kinematics, kinetics, and EMGs shows how the production of force and energy by individual muscles contribute to the energy flow among the individual segments during task execution.     

The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP

The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation. Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined. A recent study indicates that Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, raising the possibility that it might cycle between the cytoplasm and chloroplast as a soluble preprotein receptor. In the present study, we examined the mechanism of targeting and insertion of the Arabidopsis thaliana orthologue of Toc159, atToc159, to chloroplasts. Targeting of atToc159 to the outer envelope membrane is strictly dependent only on guanine nucleotides. Although GTP is not required for initial binding, the productive insertion and assembly of atToc159 into the Toc complex requires its intrinsic GTPase activity. Targeting is mediated by direct binding between the GTPase domain of atToc159 and the homologous GTPase domain of atToc33, the Arabidopsis Toc33/34 orthologue. Our findings demonstrate a role for the coordinate action of the Toc GTPases in assembly of the functional Toc complex at the chloroplast outer envelope membrane.     

Cytokine regulation of periportal fibrosis in humans infected with Schistosoma mansoni: IFN-gamma is associated with protection against fibrosis and TNF-alpha with aggravation of disease

Hepatic periportal fibrosis, which affects 5-10% of subjects infected by Schistosoma mansoni, is caused by the T cell-dependent granuloma that develop around schistosome eggs. Experimental models of infection have shown that granuloma and fibrosis are tightly regulated by cytokines. However, it is unknown why advanced periportal fibrosis occurs only in certain subjects. The goal of the present study was to evaluate the cytokine response of S. mansoni-infected subjects with advanced liver disease in an attempt to relate susceptibility to periportal fibrosis with an abnormal production of cytokines that regulate granuloma and fibrosis. Fibrosis was evaluated by ultrasound on 795 inhabitants of a Sudanese village in which S. mansoni is endemic: advanced periportal fibrosis was observed in 12% of the population; 35% of the affected subjects exhibited signs of portal hypertension. Age (odds ratio (OR), 11.5), gender (OR, 4.2), and infection levels (OR, 2.2) were significantly (p < or = 0.01) associated with hepatic fibrosis. Cytokines produced by egg-stimulated blood mononuclear cells from 99 subjects were measured (75 with no or mild fibrosis; 24 subjects with advanced fibrosis). Multivariate analysis of cytokine levels showed that high IFN-gamma levels were associated with a marked reduction of the risk of fibrosis (p = 0.01; OR, 0.1); in contrast, high TNF-alpha levels were associated with an increased risk (p = 0.05; OR, 4.6) of periportal fibrosis. Moreover, infection levels were negatively associated with IFN-gamma production. These results with observations in experimental models strongly suggest that IFN-gamma plays a key role in the protection of S. mansoni-infected patients against periportal fibrosis, whereas TNF-alpha may aggravate the disease.