The lab employs a diverse set of techniques and cutting-edge approaches in cell biology, biochemistry genetics and chemistry to define the molecular mechanisms of aging-associated diseases and identify therapeutic strategies to cure these diseases. We use genetic disorders that initiate a phenotype that resembles accelerated aging
Werner Syndrome is an adult form of progeria that is characterized clinically by the premature appearance of cataracts, diabetes mellitus, neoplasia and atherosclerosis. The most common cause of death in WS individuals is myocardial infarction at the median age of 45 years. At the molecular level, cells from WS patients display a high degree of chromosomal deletions and rearrangements. The Werner syndrome protein (WRN) is a protein with exonuclease and helicase activities whose cellular function is poorly defined. Our studies revealed a functional relationship between WRN and Ku70/80, a complex that is involved in the non-homologous end joining (NHEJ) DNA repair pathway as well as in chromosome ends (telomeres) maintenance. We and others have also shown that WRN facilitates polymerization by DNA polymerase across telomeric and other GC-rich sequences in vitro. These sequences are prone to form non-canonical DNA structures that are thought to obstruct the normal progression of the replicative polymerase, suggesting that WRN may aid DNA polymerization through these sequences by unwinding secondary structures ahead of the advancing polymerase. In addition to this supporting role in DNA synthesis, our studies have identified a functional cooperativity between WRN and FEN1 in flap cleavage during strand displacement, a critical step in Okazaki fragment (OF) maturation. Remarkably, the functional interaction between WRN and FEN1 is not limited to telomeric templates, suggesting that WRN has the potential to contribute to OF processing at other genomic sites. These findings led us to suggest that WRN deficiency may result in defective OF processing in vivo, and we are currently using biochemical as well as genetic approaches to define the role of WRN in replication genomewide.
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by premature senescence. Affected children appear normal at birth, but within a year develop characteristic features of old age. The majority of HGPS children die from cardiac disease at an average age of 13 years. Genetic studies have identified a mutation in the lamin A/C gene in 18 classical HGPS cases. The mutation results in the production of a mutant lamin A protein with an internal deletion termed progerin. The mechanism by which expression of progerin leads to accelerated aging and cardiovascular disease is unknown. Lamin A is thought to be required for the maintenance of the nuclear structure and it has been proposed to influence nuclear processes such as gene transcription and DNA replication possibly through interaction with a set of yet to be identified cellular factors. We hypothesize that expression of the mutant lamin A (progerin) results in premature aging as a consequence of the altered composition and function of lamin A-containing complexes within the nucleus. Work carried out in our lab demonstrated that the abnormal lamin A aggregates accumulating in cells expressing progerin are also present in cells from normal old-age individuals but absent in cells from young-age individuals (Candelario et al. Aging Cell, 2008). We further demonstrated that accumulation of prelamin A variants that are constitutively farnesylated induce the formation of lamin A aggregates and disrupt normal nuclear morphology, leading to changes in global gene transcription that severely inhibiting cell growth (Candelario et al. Exp. Cell Res., 2011). Collectively, these results demonstrate that prelamin A metabolism is a finely poised system and small perturbations in the normal pathway that produces mature lamin A can propel cells into a dysfunctional state that might affect healthy aging.
In collaboration with the laboratory of Dr. Reddy, we are testing the molecular etiology of myotonic dystrophy type 1 and 2 (DM1 and DM2) using biochemical approaches. An RNA dominant mechanism has been shown to underlie the development of several pathological features that are common to both DM1 and DM2. Specifically mutant RNAs encoding expanded CUG and CCUG repeat sequences are known to sequester the muscleblind family of proteins to form aberrant nuclear foci. In an ongoing series of experiments we are carrying out functional study of the muscleblind proteins and are attempting to decipher the protein profile of DM nuclear foci using molecular and biochemical approaches. We have also identified transcriptome defects in DM1 myoblasts that can not be explained by MBNLs dysfunction, and demonstrated that these alterations are caused by mislocalization of the transcription factor SHARP to the cytoplasm as a consequence of expanded CUG RNA expression. These findings provide the first evidence that transcriptional misregulation contributes to DM1 pathology. More recently, we are developing high throughput screens and validation assays for small molecule therapy of triplet repeats expansion diseases.
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