Research Interests | Research Projects
The unifying theme of the research projects in my laboratory is to combine structural and biochemical studies to investigate the macromolecular structure, function, and stability of biomolecules, especially of multi-domain proteins implicated in human diseases. I am interested in studying how individual amino acids within the linear sequence of a native protein dictate its biochemical and biophysical character and how the specific structural elements within a given protein contribute to its function. My long-term scientific interest lies in exploiting this sequence-structure-function relationship in native proteins to develop strategies for the optimization of their biochemical and biophysical properties and to design enhanced proteins with specific functions.
Most of the proteins involved in important biological pathways are large multi-domain proteins, which makes it a challenge to study them at a molecular level. Therefore, in my research, I use the protein dissection methodology, where such a multi-domain protein is studied as an assembly of structurally independent, minimal functional units that can be characterized in isolation. This approach also has the advantage of yielding information about these autonomously folding isolated domains at a molecular level that can be transferred or applied to other proteins with homologous domains. In my laboratory, we routinely use a variety of biochemical tools for the in vitro solution characterization of the protein domain under investigation, as well as many biophysical and spectroscopic methods in elucidating structure, conformational change, and binding, such as florescence spectroscopy, NMR, circular dichroism, X-ray crystallography, isothermal titration calorimetry, and chromatography. We also use molecular biology to design and clone DNA constructs suitable for Ecoli. expression systems.
---------------------------------------------------------------------
The overall goal for the projects in the Vardar-Ulu laboratory is to fill the existing gaps in our understanding of the sequence-structure-function relationship for small proteins or protein domains within their relevant specific biological background and also provide new insights into our global understanding of protein folding and stability. Our current focus is on two protein domains from the Negative Regulatory Region of the multi-domain developmental protein, Notch Receptor.
Notch Biology and Domain organization of the Notch Receptor:
The integral role that Notch proteins play in the normal development and tissue homeostasis of metazoan animals is orchestrated through the tightly-regulated proteolytic processing of their heterodimerization (HD) domain that is located N-terminal to their transmembrane region. For the mammalian 300- to 350-kDa transmembrane Notch precursor, processing begins during transport to the cell surface with the cleavage of its HD domain at site S1 by a furin-like protease yielding a mature heterodimer composed of two non-covalently associated subunits Canonical Notch signaling is normally initiated when a ligand of the Delta/Serrate/Lag-2 family binds to the receptor and initiates a proteolytic cascade called regulated intramembrane proteolysis (RIP). The key proteolytic event that triggers the cascade is an ADAM-type-metalloprotease-dependent extracellular cleavage at site (S2). S2 cleavage is a necessary prerequisite for a subsequent cleavage by gamma-secretase at site S3, which permits the translocation of the intracellular domain of Notch from the cell membrane to the nucleus to activate transcription (Figure 1)[1].
There is clear evidence that Notch signaling pathway has a big role in the development of human malignancies. Mutations in Notch1 are found in T-cell acute lymphoblastic leukemia (T-ALL) [2] and aortic valve disease [3], Notch2 is prevalent in the vascular disease Alagille syndrome [4], Notch3 mutations play a significant role in the stroke causing disease CADASIL [5], while Notch4 is implicated in breast cancers [6]. Though significant progress has been made in dissecting the complex workings of this signaling pathway, there are still very limited therapeutic options available as Notch inhibitors. Thus, a molecular understanding of the Notch activation switch would greatly benefit ongoing pharmaceutical initiatives.
Figure 1. Overview of Notch signaling [7]. Ligand binding to the extracellular Notch triggers a metalloprotease cleavage at S2 site. The resulting transmembrane subunit of the receptor is a substrate for cleavage at S3 by ?-secretase, which releases the intracellular part of Notch (ICN) from the membrane. ICN migrates to the nucleus, where it assembles into a complex that turns on transcription of target genes.
Notch receptors have a modular architecture in which several repeated structural motifs, which carry out specific functions, are assembled in a highly conserved arrangement. Before ligand-induced activation, Notch is maintained in a resting, metalloprotease-resistant conformation by a conserved Negative Regulatory Region (NRR), which is sandwiched between the ligand-binding and transmembrane regions of the protein (Figure 1, yellow box). The NRR is composed of three Lin12/Notch Repeats (LNRs) and the HD domain, which contains the S1 and S2 cleavage sites. Receptors that lack the ligand binding domain are functionally inert [8-10] demonstrating that the restraints on ligand-independent signaling reside in the NRR. Conversely, truncated forms of Notch that lack the LNRs are constitutively active, suggesting that the LNRs are responsible for protecting the S2 site from activating proteases [9]. Finally, mutations in the NRRs of Notch receptors produce gain-of-function phenotypes in diverse biological contexts, including developing invertebrates [11, 12] and patients with T-ALL [2] highlighting the importance of NRR in the tight regulation of Notch signaling.
Lin12/Notch Repeats (LNRs):
The first domain we study contains the Lin12/Notch repeats (LNRs). LNRs were first described as three unique tandem modules crucial for the regulation of ligand induced proteolytic cleavage of the Notch receptor. More recently, they were identified within functionally unrelated multi-domain proteins with different domain organizations, such as pregnancy associated proteins, and stealth proteins. LNRs belong to a subset of small disulphide-rich protein folds (SDFs), which are among the most frequently utilized structural units in all of biology. However, LNRs are functionally different from most other SDFs since they participate in intramolecular regulation rather than intermolecular recognition.
Based on the existing biochemical and structural information of a prototype LNR (Figure 2) and the protein database sequence alignment predictions, each LNR contains ~35-40 residues, has at least two disulphide bonds, and an absolute requirement for a Ca2+ ion to fold into its native structure with a very small amount of regular secondary structure.
Figure 2: NMR solution structure of LNRA from human Notch1 [13].
The three main ongoing projects involving LNRs in our lab and the questions they aim to answer are:
Project 1: Characterization of the metal binding affinity and selectivity of different LNRs.
- Do all LNRs bind Ca2+ and only Ca2+ and when they do how tightly?
- What is the underlying molecular basis for the inherent metal ion specificity, selectivity, and affinity for the different LNRs?
- Are the observed metal binding properties and/or requirements the same for the folding process and the post-folding binding process?
Project 2: Investigation of the impact of Ca2+ on the folding and chemical stability of the disulfide bonds in different LNRs.
- Do all LNRs require Ca2+ to form their characteristic disulfide bonding pattern?
- Is Ca2+ important only for the folding of the LNRs or does it have other critical roles for this small protein module, such as altering the redox requirements to break the disulfide bonds?
Project 3: Investigation Determination of the impact of the total number of disulfide bonds in the folding and chemical stability of the LNRs.
- Are the redox requirements same or different for the folding of LNRs that contain two vs. three disulfide bonds?
- Are there differences in the chemical stability of the folded LNRs that contain two vs. three disulfide bonds?
Heterodimerization (HD) Domain:
HD is comprised of the ~150 amino acid long extracellular part of the Notch receptor and lies C-terminal to the LNRs. Ligand binding activates Notch by initiating a proteolytic cascade starting with a metalloprotease-dependent extracellular cleavage within the HD. This cleavage is a necessary prerequisite for a subsequent cleavage by g-secretase, which permits the translocation of the intracellular domain of Notch from the cell membrane to the nucleus to activate transcription. Under normal cellular conditions, HD is protected from metalloprotease cleavage via the steric plug created by the LNRs until ligand binding.
The HD domain is comprised of a highly structured hydrophobic core formed by an intertwined network of five ?-strands and two a-helices. About 50% of human T-cell acute lymphoblastic leukemia (T-ALL) patients have mutations that map onto this core (Figure 3). These mutations results in ligand independent activation of Notch signaling and fifteen of these mutations were shown to reduce the chemical stability of the HD domain.
Figure 3. Mapping of T-ALL tumor-associated mutations onto the hN1 NRR structure. [14] (A) LNRs are shown with a semi-transparent molecular surface representation over their ribbon diagrams and the HD Domain is shown as a ribbon with the side-chains of residues mutated in T-ALL patients shown in ball-and-stick representation. Residues are colored according to mutation site: core (green), interface (orange), or partially exposed (purple). (B) Close-up view of mutations in the HD domain.
There are several ongoing projects in the lab that study the molecular details within the HD and aim to answer the following questions using a combination of physicochemical analysis and computational techniques:
- Are there significant correlations between evolutionarily conserved amino acids and their physicochemical properties?
- Which of the conserved residues are critical in ensuring the alpha/beta structure and stability of the HD domain?
- Which amino acid substitutions cause aberrations in receptor functionality?
---------------------------------------------------------------------
Bibliography:
- Lai, E.C., Notch signaling: control of cell communication and cell fate. Development, 2004. 131(5): p. 965-73.
- Weng, A.P., et al., Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science, 2004. 306(5694): p. 269-71.
- Garg, V., et al., Mutations in NOTCH1 cause aortic valve disease. Nature, 2005. 437(7056): p. 270-4.
- McDaniell, R., et al., NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway. Am J Hum Genet, 2006. 79(1): p. 169-73.
- Fre, S., et al., Notch signals control the fate of immature progenitor cells in the intestine. Nature, 2005. 435(7044): p. 964-8.
- Uyttendaele, H., et al., Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development, 1996. 122(7): p. 2251-9.
- Gordon, W.R., et al., Structural basis for autoinhibition of Notch. Nat Struct Mol Biol, 2007. 14(4): p. 295-300.
- Kopan, R., et al., Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain. Proc Natl Acad Sci U S A, 1996. 93(4): p. 1683-8.
- Sanchez-Irizarry, C., et al., Notch subunit heterodimerization and prevention of ligand-independent proteolytic activation depend, respectively, on a novel domain and the LNR repeats. Mol Cell Biol, 2004. 24(21): p. 9265-73.
- Schroeter, E.H., J.A. Kisslinger, and R. Kopan, Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature, 1998. 393(6683): p. 382-6.
- Berry, L.W., B. Westlund, and T. Schedl, Germ-line tumor formation caused by activation of glp-1, a Caenorhabditis elegans member of the Notch family of receptors. Development, 1997. 124(4): p. 925-36.
- Greenwald, I. and G. Seydoux, Analysis of gain-of-function mutations of the lin-12 gene of Caenorhabditis elegans. Nature, 1990. 346(6280): p. 197-9.
- Vardar, D., et al., Nuclear magnetic resonance structure of a prototype Lin12-Notch repeat module from human Notch1. Biochemistry, 2003. 42(23): p. 7061-7.
- Gordon, W.R., et al., Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL. Blood, 2009. 113(18): p. 4381-90.