Derhsing Lai | PubMed Health

PubMed2005

Mutational analysis reveals an essential role for the LXXLL motif in the transformation function of the human herpesvirus-8 oncoprotein, kaposin.

Human herpesvirus-8 (HHV-8) is causally linked to Kaposi's sarcoma (KS). Sequence analysis of the genome and subsequent studies revealed several genes including kaposin, with transformation properties in cell culture. In this study, we have analyzed the requirement of Kaposin A for cellular transformation in an effort to understand its contribution towards KS pathogenesis. Comparative analysis of Kaposin with other proteins identified the LXXLL motif spanning from residues 31-35 (LVCLL). The observation that the LXXLL motif is present in nuclear receptor coactivators that mediate the interaction of coactivators with nuclear receptors has prompted us to investigate the relevance of this motif for Kaposin's function(s). Kaposin A coding sequences were cloned into a eukaryotic expression plasmid with the Flag (FL) epitope fused in-frame at the C-terminus (Kap-FL). To evaluate the role of leucine residues in the motif, site-directed mutagenesis was utilized, whereby alanine was substituted for the leucine residues (Kap-AXXAA-FL). Both Kap-FL and Kap- AXXAA-FL exhibited similar levels of expression in cells. Interestingly, the Kap-AXXAA-FL mutant failed to show transforming activity by two independent assays: anchorage-independent growth, and focus formation. Immunofluorescence (IFA) and FACS analysis indicated that Kap-FL was localized around the nucleus and at the cell surface, respectively. However, Kap-AXXAA-FL exhibited diffuse cytoplasmic staining as measured by IFA yet was still detectable on the cell surface by FACS. Ironically, both Kap-FL and Kap-AXXAAFL were able to activate the AP-1 promoter. These results support an important role for the LXXLL motif in the ability of Kaposin to induce transformation.

Amino Acid Motifs↗

PubMed2002

Remune. Immune Response.

The Immune Response Corp (IRC) is developing Remune, a potential HIV therapeutic vaccine. Remune is based on the Salk Immunogen, which is derived from an HIV isolate which has been inactivated by chemical depletion of glycoprotein 120 (gp120). Preliminary data suggested that Remune, in combination with antiviral drug therapy, results in undetectable levels of HIV. Phase III trials commenced in May 1997 and it was initially expected that registration filings would be made in 1999. However, following interim analysis of the 2500-patient, multicenter, double-blind, pivotal phase III study (study 806) in May 1999, an independent panel recommended concluding the clinical endpoint trial and IRC and licensee, Agouron, decided to pursue alternative regulatory strategies, including initiating two additional phase III surrogate marker trials. Despite this, Agouron gave IRC notice of termination of its continued development in July 2001. In August 2001, IRC informed Agouron that, due to the total number of endpoints to date falling short of that previously assumed by Agouron, it did not intend to continue Agouron's Study 202 of Remune. In July 2001, licensee Trinity Medical Group filed an NDA with the governing health authorities in Thailand for Remune. The Thai FDA certified Immune Response's Remune manufacturing facility as being in compliance with GMP standards, following an on site inspection by Thai officials in November 2001 that was performed as a requirement of Trinity's Thai NDA. As a result of this certification, Trinity expected that a "timely determination" could be made by the Thai FDA. Rhĵne-Poulenc Rorer discontinued its part in the development of Remune, with all manufacturing, marketing and distribution rights reverting to IRC. After Agouron returned rights to Remune in July 2001, IRC heldfull rights in the US, Europe and Japan, while collaborating with its partners Trinity Medical Group and Roemmers Laboratory in the Southeast Asian and Latin American markets, respectively. In June 1998, Merrill Lynch had expected an FDA filing for Remune by the end of 1999, and in May 1999 Agouron was hoping to file an NDA for the use of Remune against AIDS during 2001. Analysts at Lehman Brothers predicted in September 2001, that the product would be launched onto the market with sales of US$150 million in 2004.

AIDS Vaccines↗