I received my Ph.D. from Baylor College of Medicine in Houston, TX in the Department of Cell Biology. After graduating from Baylor, I joined the laboratory of Dr. E.J. Benz, Jr., in the Division of Hematology, at Johns Hopkins University, School of Medicine in 1998, to study protein 4.1R, a peripheral membrane protein that was originally discovered in red blood cells, in skeletal muscle. In 2001, I moved to the laboratory of Dr. R.J. Bloch, in the Department of Physiology, at the University of Maryland, School of Medicine, as an Academic Fellow, to study a small form of ankyrin 1 (small ankyrin 1) and its ligands in skeletal muscle. In 2002, I was promoted to Research Associate, and in July of 2003 to Assistant Professor. My research has been funded by grants from the Muscular Dystrophy Association and the National Institutes of Health.
My research focuses on the role of cytoskeletal proteins in muscle organization. The identification and functional characterization of molecules that form the non-contractile cytoskeleton, which in turn "build" the contractile apparatus of striated muscle, is crucial for understanding how myofibers develop, generate and transmit force, and maintain their structural integrity during contraction and relaxation. Moreover, elucidation of the fundamental biology and pathophysiology of muscle cytoskeleton will give a better insight on the etiologies that underlie severe muscular dystrophies and cardiac disorders. This will ultimately facilitate the development of innovative therapeutic strategies.
Molecular and Functional Characterization of the Giant Myofibrillar Protein Obscurin in Striated Muscle
Obscurin (~800 kDa) is the third giant protein of the contractile apparatus identified in vertebrate striated muscle, along with titin (2-4 MDa) and nebulin (~800 kDa). Like its predecessors, obscurin is a multidomain protein composed of adhesion modules and signaling domains arranged mostly in tandem. Specifically, its NH2-terminus contains 54 Immunoglobulin-C2 (Ig-C2) and 2 Fibronectin-III (Fn-III) domains, followed by an IQ motif and a conserved SH3 domain adjacent to Rho-guanine nucleotide exchange factor (Rho-GEF) and pleckstrin homology (PH) domains (Fig. 1). The COOH-terminal end of the protein consists of 2 additional Ig domains followed by a non-modular region of ~420 amino acid residues that contains several copies of a consensus phosphorylation motif for ERK kinases, similar to that found in the NH2-terminal region of titin. The obscurin gene, obscurin-MLCK, also encodes two ser/thr kinase domains, but these are apparently not expressed as part of the ~800 kDa form of the protein, and instead are made as smaller, alternatively spliced products, mainly in heart.
Unlike titin and nebulin, which are integral components of sarcomeres, obscurin is not present within sarcomeres but intimately surrounds them (Fig. 2), primarily at the level of the Z-disk and M-line (Fig. 3), where it is appropriately positioned to participate in their assembly and integration with other sarcoplasmic elements. Consistent with this, obscurin interacts with diverse protein partners located in distinct compartments within the cell, including small ankyrin 1, an integral component of the sarcoplasmic reticulum (SR) membranes, as well as titin, sarcomeric myosin and MyBP-C of the sarcomeric cytoskeleton. Given its ability to associate tightly, selectively and periodically with the periphery of the myofibril and with thick filaments, obscurin is ideally suited to coordinate the assembly and organization of the SR with myofibrillar elements in the middle of the sarcomere.
Figure 2 Figure 3
Small Ankyrin 1: An Anchor between the Sarcoplasmic Reticulum and the Myofibrillar Cytoskeleton
Ankyrins are a family of proteins that bind diverse integral membrane proteins as well as cytoskeletal components, especially spectrins. Mammals have three distinct ankyrin genes, ANK1, ANK2 and ANK3 that produce a variety of tissue specific and developmentally regulated products. In striated muscle, the products of the Ank1 gene include the large (~210 kDa) and small (~17-19 kDa) ankyrin isoforms. The NH2-terminal portion of small Ank1 (sAnk1) contains a unique 73-amino acid segment, whereas the COOH-terminal 82 residues are identical to the COOH-terminal sequence of the large, ~210 kDa ankyrin 1. The first 29 residues of sAnk1 contain a highly hydrophobic, transmembrane sequence that targets it to the network sarcoplasmic reticulum, whereas the remaining 126 amino acids extend into the myoplasm. Recent studies have postulated a key role for sAnk1 in the assembly, stabilization and sarcomeric alignment of the sarcoplasmic reticulum membranes through its association with the giant myofibrillar proteins obscurin and titin.
HAX-1: an Anti-apoptotic Protein with Dual Localization in the Muscle Cell
HS-1 associated protein X-1 (HAX-1) is a ~32 kDa protein that was originally identified in a yeast two-hybrid screen on the basis of its binding to the hematopoietic cell specific protein 1 (HS-1). An NH2-terminal Bcl-Homology domain 1 (BH1) followed by a Bcl-Homology domain 2 (BH2), a PEST motif and a COOH-terminal transmembrane (TM) domain make up its structure (Fig. 4). HAX-1 is ubiquitously expressed among different tissues, although its relevant abundance varies.
Work from our laboratory and others has demonstrated that overexpression of HAX-1 significantly improves the ability of cells to resist hypoxia/reoxygenation-induced apoptosis in the culture setting, by blocking the activation of the initiator caspase-9 and the downstream death caspase-3. Under physiological conditions, HAX-1 is predominantly localized at mitochondria and to a lesser extent at the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) membranes. Consistent with this, recombinant HAX-1 preferentially accumulates at mitochondria in HEK-293 cells, but when co-expressed with phospolamban, PLN, an SR protein that is a direct binding partner of HAX-1, it massively translocates to the ER. More importantly, the presence of PLN significantly improves HAX-1's protective effects against oxidative stress, suggesting that redistribution of HAX-1 from the mitochondria to the ER/SR membranes enhances its anti-apoptotic properties.
Lab Techniques and Equipment:
Molecular cloning, RT-PCR/Quantitative RT-PCR/Long Range RT-PCR/RACE-PCR, yeast two-hybrid screening, protein expression and purification, overexpression experiments via adenoviral-mediated gene delivery, small inhibitory RNA technology, immunofluorescent combined with confocal laser scanning microscopy, immunoelectron microscopy, tissue culture of skeletal and cardiac myotubes, cryosectioning of muscle tissue, in vitro binding assays, surface plasmon resonance, cDNA microarrays, mass spectrometry, generation of null and transgenic animal models, and in vivo transplantation experiments.
Postdoctoral Position Available:
A postdoctoral position is available immediately in Dr. Kontrogianni's laboratory to study the role of cytoskeletal proteins in muscle organization and activity. A variety of experimental approaches will be employed in these studies, including molecular (e.g., cloning, yeast-two-hybrid, RT-PCR/qRT-PCR, mutagenesis, siRNA, transgenic and knockdown models), cellular (tissue culture of skeletal and cardiac myotubes, cryosectioning of muscle tissue, immunocytochemistry combined with confocal microscopy and transfection/transduction), biochemical (surface plasmon resonance, in vitro binding assays, mass spectrometry) and genomic (cDNA and exon microarrays). State-of-the-art equipment and facilities are available. Experience in the techniques is highly desirable.
The postdoctoral fellow will be expected to be current in the literature in the field, to plan and conduct experiments, interpret data, and publish results. Applicants must possess a PhD degree (or equivalent), should have excellent oral and written communication skills, and display initiative as well as independence. Salary is based upon experience and NIH salary levels. Please send a statement of research interests and previous experience, curriculum vitae, and the names and contact information of three references to: email@example.com.