Warren D. Kruger, PhD
Office Phone: 215-728-3030
Studies on CBS
CBS is a key enzyme in the metabolism of total homocysteine (tHcy). Individuals with mutations in the CBS gene have clinical CBS deficiency, the most common inherited disorder in methionine metabolism. Most CBS mutations are single nucleotide changes that result in single amino acid substitutions in the CBS protein that cause the mutant protein to misfold and be functionally impaired. Molecular chaperones are proteins that help mediate proper protein folding in vivo. A major focus of our lab is based on our discovery that chemicals or mutations that effect the molecular chaperone environment of a cell can restore function to mutant CBS protein. We found that that it is possible to dramatically restore protein stability and enzymatic function to mutant CBS protein by treatments that either elevate Hsp70 levels, or decrease Hsp26 levels in S. cerevisiae (Figure 2). We hypothesize that chaperone proteins interact with mutant CBS protein and form a multi-protein complex called a refoldosome. Depending on the exact protein make-up of this complex, the mutant CBS protein is either refolded to a functional form, held by the complex in a stable but not functional form, or directed to the proteosome for degradation. Our goals are: 1) to determine the molecular nature of the refoldosome, 2) identify the structural elements that determine whether a mutant protein can be successfully refolded and 3) translate this work into mammalian systems.
A second CBS-related project involves the characterization of mouse models of severe CBS deficiency (classical homocystinuria). In humans, individuals with severe CBS deficiency have extremely elevated total plasma homocysteine (tHcy>200 M) and have a variety clinical symptoms including greatly elevated risk of thrombosis, osteoporosis, dislocated lenses, and mental retardation. We have created a mouse model for severe CBS deficiency by expressing a patient derived mutant human CBS transgene in a mouse deleted for endogenous mouse CBS. These mice have a variety of phenotypes including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in lifespan (Figure 3). The main goal of this research is to determine the mechanism by which loss of CBS causes these phenotypes. Our working hypothesis is that some of the phenotypes are caused by the toxic effects of elevated tHcy, while other effects are caused by reduced production of cysteine and its downstream metabolite, glutathione.
Studies on MTAP
MTAP is a key enzyme in the methionine salvage pathway that converts the byproduct of polyamine synthesis, 5’-methylthioadenosine (MTA), into adenine and methionine. The gene for MTAP lies on human chromosome 9p21 and large deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP genes are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (MtaplacZ) have an embryonic lethal phenotype dying around day 8 post-conception. Mtap/MtaplacZ heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days (Figure 4). Autopsies on these animals reveal that they often have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and FACS analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein, consistent with the hypothesis that loss of MTAP activity is tumorigenic. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF. We are currently breeding MtaplacZ mice to other mouse genetic cancer models to determine if MTAP-loss can cooperate with other oncogenes and tumor suppressor genes to cause tumorigenesis. We have also created an isogenic set of MTAP+ and MTAP- HT1080 osteosaroma cell lines to study how MTAP-loss effects tumorigenesis at the molecular level. We find that expression of MTAP results in decreased cell invasion in vitro and decreased ability to form tumors in vivo. Using microarray technology we discovered that introduction of MTAP alters mRNA transcript levels of at least 360 genes including many genes involved in cell invasion and migration. We are currently testing the hypothesis that MTAP-loss results in elevated intracellular MTA, which in turn inhibits cellular methylation reactions, including methylation of histones which results in alterations in gene expression.
Studies on Renal Cell Carcinoma
In the United States there are over 50,000 cases of Renal Cell Carcinoma (RCC) diagnosed annually resulting in more than 13,000 deaths. It is the eighth leading cause of cancer deaths in men and the fourteenth in woman. The kidney plays a central role in the clearance of nitrogenous substances and in regulating plasma amino acid levels. RCC tumors are characterized by a variety of mutations in genes that are known to be involved in a variety of basic metabolic processes, so much so that in a recent review on the subject kidney cancer was described as “a metabolic disease”. Specific genetic alterations in RCC cells affect signaling pathways involved in energy and nutrient sensing, such Akt/mTOR and AMPK pathways. In addition, RCC cells have altered metabolism related to a metabolic shift from respiration to fermentation, the so-called Warburg. The goal of this project is to understand the amino acid defects in RCC and to use these alterations to create new therapies for this disease.