Biomedical subjects

David A Drubin

Publications and source records attributed to David A Drubin.

5 recordsLinked to original sources

PubMed2007

The chloromethylketone protease inhibitor AAPF(CMK) also targets ATP-dependent helicases and SAP-domain proteins.

We have been studying a nuclear protease, which appears to be involved in cellular transformation, as well as in infections with high-risk human papillomaviruses (HPVs). This protease has a chymotrypsin-like substrate specificity and the chloromethylketone inhibitor AAPF(CMK) is a potent (and relatively selective) inhibitor of it. Recently, we have observed that AAPF(CMK) has potent effects in some model systems which appear not to be mediated by decreases in the nuclear protease. Here we show that AAPF(CMK) selectively reacts with ATP-dependent helicases as well as a limited spectrum of proteins in other DNA repair/chromatin remodeling nuclear complexes, including for example Cohesin complex components and proteins containing SAP-domains. In vitro, AAPF(CMK) selectively reacts with SV40 large T antigen, and inhibits its helicase activity.

Adenosine Triphosphate↗

PubMed2006

Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics.

BACKGROUND: Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC) at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated. RESULTS: We have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells. CONCLUSION: Transcriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to the analysis of chromosome motion a machine visual screening approach that used unbiased visual data rather than segmented geometric data. This novel analytical approach will allow for high-throughput study of processes that can be monitored via alterations in chromosome motion and connectivity with the nuclear periphery.

Cell Survival↗

PubMed2006

A protease inhibitor specifically inhibits growth of HPV-infected keratinocytes.

Protease inhibitors have been known to exhibit anticarcinogenic activity in a variety of model systems, although the biological target(s) and mechanism remain enigmatic. Human papillomavirus (HPV) is the primary etiological agent of cervical cancer. Here we show that a nuclear chymotrypsin-like protease activity (NCLPA), which appears to be involved in transformation in several different experimental models, is significantly elevated in keratinocytes infected with high-risk HPV. Further, we demonstrate a marked growth inhibition of organotypic raft cultures, which is specific for cells infected with high-risk HPV types, using a chloromethyl ketone inhibitor previously shown to be relatively selective for the NCLPA. Surprisingly, this HPV-dependent inhibitory effect is independent of any alterations in the NCLPA. This finding has clear implications for the development of novel therapeutics specifically targeted to cervical dysplasias with HPV-infected cells.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Developmentally induced changes in transcriptional program alter spatial organization across chromosomes.

Although the spatial location of genes within the nucleus has been implicated in their transcriptional status, little is known about the dynamics of gene location that accompany large-scale changes in gene expression. The mating of haploid yeast Saccharomyces cerevisiae is accompanied by a large-scale change in transcription and developmental program. We examined changes in nuclear organization that accompany stimulus by the mating pheromone alpha factor and found that most alpha-factor-induced genes become associated with components of the nuclear envelope. The myosin-like protein Mlp1, which has been implicated in mRNA export, was further shown to exhibit RNA dependence in its association with alpha-factor-induced genes. High-resolution mapping of association of chromosome III with Mlp1 revealed alpha-factor-dependent determinants of nuclear pore association, including origins of replication, specific intergenic regions, and the 3' ends of transcriptionally activated genes. Taken together, these results reveal RNA- and DNA-dependent determinants of nuclear organization as well as a detailed picture of how an entire chromosome alters its spatial conformation in response to a developmental cue.

Chromosomes, Fungal↗

PubMed2004

Spontaneous transformation of an immortalized hepatocyte cell line: potential role of a nuclear protease.

In this study, we utilized an in vitro model of spontaneous transformation/progression, an SV40 large T antigen-immortalized rat hepatocyte cell line (designated CWSV14) that is very weakly tumorigenic at low-passage, but acquires a transformed phenotype upon extended passage in cell culture. Here we show that this mid-passage transformation is accompanied by development of aneuploidy and disorganization of the actin cytoskeleton, concomitant with a large increase in a chymotrypsin-like nuclear protease activity which we have previously implicated in chemical transformation of fibroblasts and ras-transformation of hepatocytes. Passage of the CWSV14 cells with AAPF(cmk), a relatively selective inhibitor of the nuclear protease activity, abrogates the acquisition of the transformed phenotype and prevents the changes in the actin cytoskeleton. We hypothesize that the nuclear protease may play a role in initiating development of genomic instability, paralleling the archetypical role of proteases in paradigms such as the SOS-type responses in bacteria and yeast.

Animals↗