biochemical and molecular analyses of amphibian limb regeneration and natural products
	Craig Crews, Ph.D. Lewis B. Cullman Professor of Molecular, Cellular and Developmental Biology Professor of Chemistry Professor of Pharmacology Room: KBT 400 Phone: (203) 432-9364 / 6553 Email: Craig.Crews@yale.edu Lab Web site B.A. University of Virginia; Ph.D. Harvard University

Targeting Intracellular Proteins for Degradation using Small Molecules. Hela cells (top left) expressing Green Fluorescent Protein fused to the androgen receptor (AR) were incubated with 25 µM DHT-ProTac (above center), which targeted the AR fusion protein for degradation within 60 minutes (top right).

Ectopic Limb Regeneration. (top) A 14 day hind limb blastema from a pigment axolotl (Ambystoma mexicanum) grafted onto the upper leg of an albino axolotl. (bottom) Outline of PROTAC-mediated Protein Degradation

The two foci of our laboratory are 1) exploring the molecular mechanisms that underlie amphibian limb regeneration and 2) the use of small molecules to probe various questions in cell biology.

Urodele amphibians such as Mexican salamanders (aka, axolotls) have the amazing ability to regenerate missing lens, tails, jaws and limbs. This epimorphic phenomenon begins with the closure of the wound via migration of existing surrounding epidermis, which over the course of several days becomes an unique structure known as the Regenerative Epidermis (RE). This RE is required for regeneration since its removal prevents limb regrowth; however, it is unclear if the RE is playing a ‘permissive’ or a ‘inductive’ role in the generation of the undifferentiated cell mass (aka, blastema) that accumulates under the RE. We have recently identified 124 genes that are expressed 1.5x more highly in the RE relative to non-regenerating, wound healing epidermis. We are currently exploring several of these secreted and membrane bound proteins to determine if they play an inductive role in cellular dedifferentiation (i.e., blastema formation). If so, this induction of adult dedifferentiated cells could be considered a natural analogy to iPS cell generation via the use of exogenously supplied genes.

In addition to our interest in regenerative medicine, we are also developing novel reagents, which will allow us to explore new areas in cell biology. This 'chemical genetic' approach uses biologically active natural products and de novo designed small molecules to identify critical components in intracellular processes. In the past few years, our efforts have focused on anti-angiogenic, antitumor, and anti-inflammatory natural products. More recently, we have explored the use of small molecules to control intracellular protein levels, either by inhibiting their degradation or the induction of their proteolysis via the 26S proteasome. A goal of this research is to develop novel methodologies that would allow for small molecule control of the ‘undruggable proteome’.

Selected Publications

Leuenroth, SJ , CM Crews. (2008) Triptolide Induced Transcriptional Arrest is Associated with Changes in Nuclear Sub-Structure Cancer Research 68:5257-5266.

Ju R, Cirone P, Lin S, Griesbach H, Slusarski DC and CM Crews. (2010)Activation of the Planar Cell Polarity Formin DAAM1 Leads to Inhibition of Endothelial Cell Proliferation, Migration and Angiogenesis, Proc. Nat. Acad. Sci, 107(15):6906-11.

Campbell LJ, Suárez-Castillo EC, Ortiz-Zuazaga H, Knapp D, Tanaka EM, CM Crews. (2011) Gene expression profile of the regeneration epithelium during axolotl limb regeneration. Developmental Dynamics. 2011 240 (7):1826-40.

Fedeles SV, Tian X, Gallagher AR, Mitobe M, Nishio S, Lee SH, Cai Y, Geng L, CM Crews, Somlo S. A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation. Nature Genetics 43(7):639-47.

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