Showing papers by "Jun Komano published in 2007"

Separate elements are required for ligand-dependent and -independent internalization of metastatic potentiator CXCR4

Abstract: The C-terminal cytoplasmic domain of the metastatic potentiator CXCR4 regulates its function and spatiotemporal expression. However, little is known about the mechanism underlying constitutive internalization of CXCR4 compared to internalization mediated by its ligand, stromal cell-derived factor-1 alpha (SDF-1α)/CXCL12. We established a system to analyze the role of the CXCR4 cytoplasmic tail in steady-state internalization using the NP2 cell line, which lacks endogenous CXCR4 and SDF-1α. Deleting more than six amino acids from the C-terminus dramatically reduced constitutive internalization of CXCR4. Alanine substitution mutations revealed that three of those amino acids Ser344 Glu345 Ser346 are essential for efficient steady-state internalization of CXCR4. Mutating Glu345 to Asp did not disrupt internalization, suggesting that the steady-state internalization motif is S(E/D)S. When responses to SDF-1α were tested, cells expressing CXCR4 mutants lacking the C-terminal 10, 14, 22, 31 or 44 amino acids did not show downregulation of cell surface CXCR4 or the cell migration induced by SDF-1α. Interestingly, however, we identified two mutants, one with E344A mutation and the other lacking the C-terminal 17 amino acids, that were defective in constitutive internalization but competent in ligand-promoted internalization and cell migration. These data demonstrate that ligand-dependent and -independent internalization is genetically separable and that, between amino acids 336 and 342, there is a negative regulatory element for ligand-promoted internalization. Potential involvement of this novel motif in cancer metastasis and other CXCR4-associated disorders such as warts, hypogammaglobulinemia, infections and myelokathexis (WHIM) syndrome is discussed. (Cancer Sci 2007; 98: 373–379)

Inhibiting lentiviral replication by HEXIM1, a cellular negative regulator of the CDK9/cyclin T complex.

Abstract: Objective Tat-dependent transcriptional elongation is crucial for the replication of HIV-1 and depends on positive transcription elongation factor b complex (P-TEFb), composed of cyclin dependent kinase 9 (CDK9) and cyclin T. Hexamethylene bisacetamide-induced protein 1 (HEXIM1) inhibits P-TEFb in cooperation with 7SK RNA, but direct evidence that this inhibition limits the replication of HIV-1 has been lacking. In the present study we examined whether the expression of FLAG-tagged HEXIM1 (HEXIM1-f) affected lentiviral replication in human T cell lines. Methods HEXIM1-f was introduced to five human T cell lines, relevant host for HIV-1, by murine leukemia virus vector and cells expressing HEXIM1-f were collected by fluorescence activated cell sorter. The lentiviral replication kinetics in HEXIM1-f-expressing cells was compared with that in green fluorescent protein (GFP)-expressing cells. Results HIV-1 and simian immunodeficiency virus replicated less efficiently in HEXIM1-f-expressing cells than in GFP-expressing cells of the five T cell lines tested. The viral revertants were not immediately selected in culture. In contrast, the replication of vaccinia virus, adenovirus, and herpes simplex virus type 1 was not limited. The quantitative PCR analyses revealed that the early phase of viral life cycle was not blocked by HEXIM1. On the other hand, Tat-dependent transcription in HEXIM1-f-expressing cells was substantially repressed as compared with that in GFP-expressing cells. Conclusion These data indicate that HEXIM1 is a host factor that negatively regulates lentiviral replication specifically. Elucidating the regulatory mechanism of HEXIM1 might lead to ways to control lentiviral replication.

In vitro translation to study HIV protease activity

Abstract: HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for virus replication. Here, we describe an in vitro transcription/translation-based method of phenotyping HIV-1 PR. In this system, both substrate and PR for the assay can be prepared by in vitro transcription/translation. Protease activity is estimated by the cleavage of a substrate, as measured by enzyme-linked immunosorbent assay (ELISA). This assay is safe, rapid, and requires no special facility to be carried out. Our rapid phenotyping method of HIV-1 PR may help evaluate drug resistance, useful when choosing an appropriate therapeutic regiment, and could potentially facilitate the discovery of new drugs effective against HIV-1 PR.