Research Accomplishments

Identification of subunit configurations of the receptors for insulin and the insulin-like growth factors 

Prior to the 1980s, there was an appreciation that receptor proteins for these peptides were present on cell surface membranes, but there was no biochemical information on these receptors. To approach this problem, Paul Pilch, then a postdoc in our lab, synthesized disuccinimidyl suberate as an affinity-labeling reagent for peptide hormone receptors and identified the two insulin receptor subunits, denoting these as alpha and beta in several publications. This approach was then used to deduce the disulfide-linked subunit configurations of the receptors for insulin and the insulin-like growth factors (IGF). This work clarified the overlapping binding affinities of insulin and the IGF ligands for these receptors and revealed that the IGF-2 receptor does not mediate the major bio-effects of these peptides. We showed the IGF-2 receptor is instead a target of insulin signaling, cloned the IGF-2 receptor in collaboration with Axel Ullrich and reported in Science it's identical to the mannose-6-phosphate receptor, i.e., it's a bi-functional receptor. 

Pilch, P.F. and Czech, M.P. (1979). Interaction of Cross-Linking Agents with the Insulin Effector System of Isolated Fat Cells.  Covalent Linkage of 125I-Insulin to a Plasma Membrane Receptor Protein of 140,000 Daltons. J. Biol. Chem., 254: 3375-3381.

Massague, J and Czech, M.P. (1982). The Subunit Structures of Two Distinct Receptors for Insulin-Like Growth Factors I and II and Their Relationship to the Insulin Receptor.  J. Biol. Chem., 257: 5038-5045.

MacDonald. R.G., Pfeffer, S.R., Coussens, L., Tepper, M.A., Brocklebank, C.M., Mole, J.E., Anderson, J.K., Chen, E., Czech, M.P. and Ullrich, A. (1988) A Single Receptor Binds both Insulin-like Growth Factor II and Mannose-6-Phosphate.  Science, 239: 1134-1137.

Identification of a key downstream target of insulin-stimulated PIP3 generation

As several groups identified PI 3-kinase as a component in insulin signaling, it became important to identify downstream signaling elements. Our group established an expression cloning screen for identifying targets of the PI 3-kinase pathway in insulin and IGF-1 receptor signaling, and discovered Grp1, a novel downstream effector of the signaling lipid PtdIns(3,4,5)P3 (PIP3). We reported in Science the identification of Grp1, showing that it defines a novel PI 3-kinase-mediated signaling pathway distinct from Akt, linking PIP3 to the activation of ArfGTPases. Jes Klarlund in our lab showed the Grp1 PH domain has the highest specificity for PIP3 of all the PH domains studied. The structural basis of selective PIP3 binding to the crystallized PH domain of Grp1 was solved in collaboration with David Lambright, published in Molecular Cell.  The Grp1 PH domain fused to GFP is also now  widely used by researchers as a unique  reagent  to define the generation of PIP3 at the plasma membrane.

Klarlund, J., Guilherme, A., Holik, J.J., Virbasius, J.V., Chawla, A., Czech, M.P. (1997). Signaling by3,4,5-Phosphoinositide   through   Proteins   Containing   Pleckstrin   and   Sec7   Homology   Domains. Science, 275(5308):1927-30. 

Lietzke, S.E., Bose, S., Cronin, T., Klarlund, J., Chawla, A., Czech, M.P., Lambright, D.G. (2000). Structural Basis of 3-Phosphoinositide Recognition by Pleckstrin Homology Domains. Molecular Cell, 6:385-394. 
 

Identification of lipid droplet proteins and mechanisms of lipid storage

A focal point of our lab over many years has been the mechanisms that control triglyceride storage in adipocytes and their relationships to insulin resistance. We discovered a family of Cide-domain containing proteins is associated with lipid droplets in adipocytes and hepatocytes, and regulate lipid storage and turnover in these metabolic cell types. Gene deletion of Cidec/FSP27 and Cidea in mice has corroborated their key roles in lipid metabolism and whole body energy expenditure, and a human subject with a disrupting mutation in CIDEC displays lipodystrophy, insulin resistance and type 2 diabetes. We also found evidence that decreased lipid droplet protein expression in human adipose tissue may correlate with the appearance of insulin resistance, consistent with the hypothesis that they help sequester neutral lipids within adipocytes to protect other tissues from lipotoxicity. 

Puri V, Konda S, Ranjit S, Aouadi M, Chawla A, Chouinard M, Chakladar A, Czech M.P. (2007) Fat- specific protein 27, a novel lipid droplet protein that enhances triglyceride storage. J Biol Chem., 282(47):34213-8..

Puri, V., Ranjit, S., Konda, S. Nicoloro, S.M., Straubhaar J, Chawla, A., Chouinard, M., Lin, C., Burkart A, Corvera, S., Perugini, R.A., Czech, M.P. (2008) Cidea is associated with lipid droplets and insulin sensitivity in humans. Proc. Nat. Acad. Sci. USA, 105(22): 7833-7838. 

Therapeutic strategies leveraging the power of siRNA and CRISPR technologies

Our laboratory has developed new modes of delivery of siRNA and CRISPR components to metabolic tissues for the purpose of targeting gene pathways in metabolic disease. We’ve used siRNA screening techniques to discover genes encoding metabolic suppressor activities, e.g., RIP140, that are now being advanced as potential therapeutic targets for obesity and type 2 diabetes. In collaboration with the Ostroff laboratory, vehicles for delivery of siRNAs to adipose tissue and liver macrophages were developed, and in collaboration with the Khvorova and Watts laboratories self-delivery, fully chemically modified siRNAs are being developed for metabolic tissue targeting. In addition, recent experiments using CRISPR techniques to disrupt RIP140 in human adipocytes in collaboration with the Corvera laboratory have yielded a promising strategy advancing toward cell therapeutic applications. Overall, these various interactive projects have leveraged the high potential of siRNA and CRISPR as potential future therapeutics for applications in obesity and type 2 diabetes.

Powelka AM, Seth, A, Virbasius, JV, Kiskinis, E, Nicoloro, SM, Tang, X, Straubhaar, J, Cherniack, AD, Parker, MG, Czech, M.P. (2006) Suppression of oxidative metabolism and mitochondrial biogenesis by the transcriptional corepressor RIP140 in mouse adipocytes. Journal of Clinical Investigation, 116(1):125-136.

Jourdan T, Godlewski G, Cinar R, Bertola A, Szanda G, Liu J, Tam J, Han T, Mukhopadhyay B, Skarulis MC, Ju C, Aouadi M, Czech M.P., Kunos G. (2013) Activation of the Nlrp3 inflammasome in infiltrating macrophages by endocannabinoids mediates beta cell loss in type 2 diabetes. Nature Medicine, 19(9):1132-1140.

Yenilmez, B., Wetoska, N., Kelly, M., Echeverria, D., Min, K., Lifshitz, L., Alterman, J.F., Hassler, M.R., Hildebrand, S., DiMarzio, C., McHugh, N., Vaneieli, L., Sousa, J., Pan, M., HanX., Brehm, M.A., Khvorova, A., Czech, MP. (2022) An RNAi therapeutic targeting hepatic DGAT2 in a genetically obese mouse model of nonalcoholic steatohepatitis. Molecular Therapy, 30 (3), 1329-1342 

Tsagkaraki, E, Nicoloro, S.M., DeSouza, T., Solivan-Rivera, J., Desai, A., Lifshitz, L.M., Shen, Y., Kelly, M., Guilherme, A., Henriques, F.,et al Czech, M.P., (2021) CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease. Nature Communications, 12 (1):6931-6947