CCL20 is overexpressed in Mycobacterium tuberculosis-infected monocytes and inhibits the production of reactive oxygen species (ROS).

CCL20 is a chemokine that attracts immature dendritic cells. We show that monocytes, cells characteristic of the innate immune response, infected with Mycobacterium tuberculosis express the CCL20 gene at a much higher level than the same cells infected with non-tuberculous mycobacteria. Interferon (IFN)-γ, a fundamental cytokine in the immune response to tuberculosis, strongly inhibits both the transcription and the translation of CCL20. We have also confirmed that dendritic cells are a suitable host for mycobacteria proliferation, although CCL20 does not seem to influence their intracellular multiplication rate. The chemokine, however, down-regulates the characteristic production of reactive oxygen species (ROS) induced by M. tuberculosis in monocytes, which may affect the activity of the cells. Apoptosis mediated by the mycobacteria, possibly ROS-dependent, was also inhibited by CCL20.

In vivo monitoring of intracellular ATP levels in Leishmania donovani promastigotes as a rapid method to screen drugs targeting bioenergetic metabolism.

A method for the rapid screening of drugs targeting the bioenergetic metabolism of Leishmania spp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels of Leishmania donovani promastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form of Phothinus pyralis luciferase and incubated with free-membrane permeable D-luciferin analogue D-luciferin-[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (DeltaPsi(m)) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 microM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity.

Acidic pH stress induces protein tyrosine phosphorylation in Leishmania pifanoi.

In order to determine whether in vitro Leishmania exposure to conditions comparable to those encountered inside the host cell would induce specific signals, we have studied tyrosine phosphorylation patterns in Leishmania pifanoi. Incubation of L. pifanoi at acidic pH resulted in the phosphorylation of several proteins including three of 27, 43 and 51 kDa, as well as the dephosphorylation of a 175 and a 39 kDa proteins in promastigotes recently transformed. In contrast, heat shock at 37 degrees C did not change the tyrosine phosphorylation pattern. Phosphorylation only occurs at pH 5.0 or lower and reached completion after 1 h. Changes returned to the initial conditions in 2 h after pH medium neutralization, indicating a reversible mechanism of phosphorylation.

Mutations in the non-catalytic domains of Fyn and Fgr tyrosine kinases reveal differences in mechanisms of their regulation.

Non-catalytic domains of tyrosine kinases from the Src family are believed to be regulated intra- and intermolecularly through protein-protein interactions. We have deleted the SH2 and SH3 domains from Fyn and Fgr and have generated two point mutations in residues completely conserved in all members of the Src family. The dramatically different biological effects of these mutations suggest that non-catalytic domains regulate Src family kinase activities through distinctly different mechanisms.

Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains.

In the second of a series of experiments designed to identify p47nck-Src homology 3 (SH3)-binding molecules, we report the cloning of SAKAP II (Src A box Nck-associated protein II) from an HL60 cDNA expression library. This molecule has been identified as a cDNA encoding the protein product of WASP, which is mutated in Wiskott-Aldrich syndrome patients. Studies in vivo and in vitro demonstrated a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antibodies recognized a protein of 66 kDa by Western blot (immunoblot) analysis. In vitro translation studies identified the 66-kDa protein as the protein product of WASP, and subcellular fractionation experiments showed that p66WASP is mainly present in the cytosol fraction, although significant amounts are also present in membrane and nuclear fractions. The main p47nck region implicated in the association with p66WASP was found to be the carboxy-terminal SH3 domain.

Cloning and characterization of cbl-b: a SH3 binding protein with homology to the c-cbl proto-oncogene.

We have cloned a new gene, cbl-b, with homology to the c-cbl proto-oncogene. A large protein is predicted (approx. MW 108,000) that has a proline rich domain, a nuclear localization signal, a C3HC4 zinc finger and a putative leucine zipper. There is striking nucleotide and amino acid homology to the c-cbl proto-oncogene most notably in the structural motifs described above. Cbl-b is expressed in normal and malignant mammary epithelial cells, in a variety of normal tissues, and in hematopoietic tissue and cell lines. Cbl-b expressions is up-regulated with macrophage/monocyte differentiation of the HL60 and U937 cell lines. There is direct association of the cbl-b protein with the Src Homology 3 domains of several proteins including signaling, cytoskeletal and adaptor proteins. Our data suggest that cbl-b encodes a protein which can interact with signal transduction proteins to regulate their function or to be regulated by them. Together, cbl-b and c-cbl are members of a novel family of proto-oncogenes involved in signal transduction.

Identification of the major tyrosine kinase substrate in signaling complexes formed after engagement of Fc gamma receptors.

We have recently identified the protein product of the c-cbl proto-oncogene as an SH3 binding protein expressed in macrophages. To investigate the possibility that p120c-cbl is involved in signaling pathways initiated by cell surface receptors for IgG (Fc gamma R), lysates of HL60 cells were examined for tyrosine phosphorylation of p120c-cbl upon Fc gamma R engagement. Our findings demonstrate that p120c-cbl is tyrosine-phosphorylated upon Fc gamma R engagement and that this molecule represents the major tyrosine kinase substrate in this signaling pathway. Protein complexes containing p120c-cbl, p72syk, and p56lyn were observed either in resting or activated cells. In vitro studies showed that the direct association between p120c-cbl and p56lyn was mediated by the SH3 domain of p56lyn.

Physical association between Src homology 3 elements and the protein product of the c-cbl proto-oncogene.

To investigate the nature of proteins recognized by Src homology 3 (SH3) domains, a cDNA expression library was prepared from macrophages and screened with a probe representing the three SH3 domains of p47nck. Two clones were isolated, and one, designated SAKAP I (for Src A box Nck-associated protein I), contained the carboxyl-terminal half of the cbl proto-oncogene product. Studies in vitro demonstrated reactivity between SAKAP I and SH3 domains derived from a variety of molecules. Wide variations in this assay suggested a high degree of specificity inherent in SAKAP I binding. Moreover, it was possible to demonstrate an in vivo association between p47nck and p120c-cbl in HL60 cells. These findings suggest that proteins containing SH3 elements regulate Cbl function.

Specificity of protein interactions with highly related SRC homology (SH) domains of FGR and FYN protein-tyrosine kinases.

As an approach toward identification and isolation of cellular proteins that may act as substrates or effectors of the SRC-family of protein-tyrosine kinases, fusion proteins containing noncatalytic elements of two highly related SRC-family members were tested for their ability to recognize distinct molecules present in lysates of cells known to normally express both enzymes. Our results demonstrate differences of protein binding between the SH2 elements of FYN and FGR kinases, but do not discriminate proteins binding to their SH3 domains.