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MB&B CSB YALE

Our laboratory studies the biochemical and structural basis of various disease implicated processes described below. We use a variety of techniques that include X-ray crystallography, biochemistry, molecular biology, and computational biology. We also study new X-ray crystallographic methods to facilitate the structural work.

Innate immune responses to HIV infection
Fatty acid synthesis, obesity, and cancer
Fanconi anemia and cancer
tRNA maturation and modification
X-ray crystallography at low resolution

Innate immune responses to HIV infection
The first line of defense:
When HIV enters a cell, it encounters an antiviral DNA cytidine deaminase, APOBEC3G, which induces extensive mutations in the viral DNA and renders the virus non-infectious. To evade the host defense system, HIV expresses the virion infectivity factor, Vif, which targets APOBEC3G for destruction by the proteasome.

We aim to dtermine the chemical and structural principles by which APOBEC3G deaminates viral DNA and the mechanisms by which Vif sequesters APOBEC3G. Information gained will be used to direct structure-based design of anti-HIV drugs that inhibit Vif. Screening of inhibitors will be carried out at Yale Chemical Genomics Screening Facility.

The cross-species barrier:
Retroviruses are often limited to a small number of host organisms due to species-specific cellular restriction factors. The tripartite motif protein TRIM5alpha is an important component of the cross-species barrier to HIV and many other retroviruses. TRIM5alpha inhibits retrovirus infection likely through interactions with the viral capsid protein (CA). Our goal is to characterize the TRIM5alpha-CA interaction in vitro and establish the structural basis for this interaction.

The last line of defense:
HIV completes its life cycle by budding off the host cells for the next round of infection. The host transmembrane protein BST-2/CD317 (tetherin) tethers HIV, and diverse other enveloped viruses, to infected cells preventing their release. HIV evades the host defense by the viral protein u, Vpu, which antagonizes tetherin restriction by an unknown mechanism. Our goal is to establish the chemical and structural principles by which techerin restricts HIV release and the mechanisms by which Vpu antagonizes tetherin. Information gained will be used for the design of new antiviral therapeutics.

Fatty acid synthesis, obesity, and cancer [Top]
In animals and fungi the entire metabolic pathway for the production of common fatty acids is carried out by a large cellular machine, fatty acid synthase (FAS), through a complex series of over forty reactions. Besides its important function in fat metabolism, FAS plays fundamental roles in cancer pathogenesis. An elevated level of tumor-associated FAS confers growth and survival advantages, making it a hallmark of most human cancers.

Our aim is to establish a complete structural and enzymatic framework of FAS functions and use this information for rational design of FAS inhibitors to specifically target tumors and obesity. We have determined the crystal structure of of the yeast FAS. We will continue to examine different FAS reaction states and also determine the structure of human FAS. Toward our goal we have designed a multi-disciplinary approach that incorporates a variety of newly developed biochemical and structural biology methods, high-throughput screening of synthetic and natural inhibitors, as well as novel inhibitor delivery systems.

Fanconi anemia and cancer [Top]
Fanconi anemia (FA) is a childhood disease characterized by multiple devastating symptoms, which include bone marrow failure, developmental abnormalities, and a high incidence of cancer. Recent evidence has revealed that the proteins mutated in Fanconi anemia constitute a novel DNA damage response network that incorporates breast and ovarian tumor suppressor proteins, whose genetic inactivation represents the underlying basis of certain types of FA.

Our long-term goal is to elucidate the crosstalk between the FA pathway and the DNA repair machinery that controls cancer. Toward this goal we will carry out biochemical studies of FA proteins involved in the DNA damage response, identify their interaction complexes, and determine their crystal structures. Information gained from our endeavors may lead to new medical advances that provide improved anticancer treatment to alleviate the sufferings of children with Fanconi anemia, and possibly a broader population afflicted with cancer.

tRNA maturation and modification [Top]
Post-transcriptional modifications are crucial to the function of tRNAs. Mature tRNA contains an absolutely conserved uridine at position 8 that is important for tRNA stability. This uridine is further modified to a 4-thiouridine in archaeal and bacterial tRNAs to achieve photochemical reactivity. Recent studies have discovered that most of the tRNA genes in the archaeon Methanopyrus kandleri contain a cytidine at position 8. Therfore, two important posttranscriptional modifications, cytidine deamination and uridine thiolation, must occur to produce the final 4-thiouridine at this position.

Our objective is to to establish the structural and enzymatic basis of these two important tRNA maturation processes. We will delineate the biochemical and biophysical properties of the novel cytidine deamination and uridine thiolation processes and obtain high-resolution crystal structures of the enzymes in un-liganded forms and their tRNA complexes. Information gained will also shed light on the long-sought cytidine deaminase acting on tRNA.

X-ray crystallography at low resolution: [Top]
Electron microscopy (EM) to X-ray crystallography: Low-resolution EM images can be used for X-ray structure determination by molecular replacement methods. The phases obtained can be extended to higher resolution by density modification techniques.

Electron density de-blurring: The electron density map obtained by X-ray diffraction is often blurred due to moeclular motions and crystal disorders. We study the decoupling of the blurring effect from the diffraction data by sharpening techniques that treat individual molecular domains separately.

Atomic models at very low resolution: Very low-resolution electron density maps (6-8 Å) can be used as a restraint for folding software in de novo calculations of atomic models.

last modified:2009-08-20