Aleksandar Jeremic

In cells there are hundreds of macromolecular assemblies ranging in size from a few to over one hundred million Daltons such as the nuclear pore complex. Despite progress in structural genomics and modern instrumentation, high resolution structural information is available for only a small fraction of these assembles known to play critical roles in signal transduction, genetic, and metabolic networks. Even fewer molecular structures are available for large macromolecular complexes (<100 KD) in which the atomic details of protein-protein, protein-nucleic acid or protein-lipid interactions have been elucidated. To gain new insights into the structure, organization and dynamics of these large macromolecular assemblies, my laboratory adopts nano-approach that includes high-resolution optical, force and spectral imaging, X-ray crystallography and CD/Raman/MS spectroscopy. By using ß-cell as a model system and with help of a new generation of imaging tools, we seek to elucidate general principles underlying the self-organization of complex dynamic structures inside cells. Delineating the 3-D structure and molecular architecture of these super assemblies is essential to mechanistically understand its role in mediating fundamental biological processes and diseases.

Type II Diabetes mellitus (T2DM) is a complex and heterogeneous metabolic disease that has reached epidemic proportions. The onset of T2DM is characterized by two determining factors: the insufficient ability to secrete insulin and/or the resistance to its biological action. The ability to store insulin and release it in a regulated manner in response to a complex array of physiological signals is the major feature of pancreatic islet ß-cells. Understanding the mechanism that regulates insulin secretion may allow the development of novel drug therapies with possibility to adjust secretory deficiencies. Our research goal is to elucidate more precisely the structure and organization of cellular machinery that regulates insulin release in ß-cell and to find the cause for islet amyloid fibril formation. These studies should also reveal underlying general principles of organization of supramolecular complexes and its dynamics in cells.

Aim 1: Unraveling structure and composition of membrane machinery regulating insulin release in ß-cell. Glucose-induced insulin secretion in ß-cells are biphasic process, consisting of transient and rapid first phase followed by a sustained second phase. Insulin release is tightly controlled process as only a small fraction of the ß-cell insulin content is released during secretion. Because T2DM patients have defective insulin secretion, it is important to understand the cellular mechanism underling biphasic insulin release. We integrate biochemical and biophysical methods with the high-resolution cell imaging such as atomic force microscopy (AFM) to identify structure, dynamics and organization of the insulin release machinery located at the ß-cell plasma membrane. Membrane reorganization and dynamics of insulin release in ß-cell is studied in real time through integrated optical and force microscopy (confocal/AFM imaging). In biology, the distinct advantage of this scanning probe microscope over the electron microscope (EM) is its unique ability to directly monitor changes in the conformation or aggregation state of macromolecules, and to study dynamics aspects of molecular interaction in their physiological buffer environment.

Aim 2: Elucidating the molecular mechanism of aberrant protein (amyloid) formation in the islets of Langerhans. Genetic, endocrine and environmental factors contribute to the development of diabetes. Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. Islet amyloid polypeptide (IAPP or amylin) is a 37 aa-residue peptide hormone, a protein co-expressed and secreted with insulin by islet ß-cells. In the late-onset diabetes, however, it comprises the major fibrillar component of the islet amyloid deposits, a hallmark of T2DM (found in over 90 % of patients). Diabetes is one of a family of diseases featuring fibrillar amyloid deposits, such as Alzheimer's (ß-amyloid), Huntington's disease (huntingtin) and mad-cow (prion) disease. Intervention to control fibril growth and aggregation has great therapeutic potential, but it requires a rational understanding of the molecular mechanisms governing fibril assembly and its dynamics in cells. Therefore, by using AFM in our studies, it is possible to reveal conditions in the cell that enhance or reduce fibril formation. For example, different reagents can be added to the cultured human ß-cells or islets while fibrils form, and thus their potential as activators or inhibitors of fibril assembly could be directly (in situ) investigated at the single cell and fibril level. Combination of different methodologies and techniques, such as intracellular Ca2+ imaging (to study amyloid-induced cell damage), ELISA and/or confocal microscopy (to study the cell functionality, i.e. assessment of ß-cell to release insulin), photon correlation spectroscopy (to observe formation of amyloid fibrils and their intermediates), CD and Raman spectroscopy (to study peptide conformational transitions), and the high-resolution imaging of fibril structures by AFM, will bring us a step closer to the goal of finding the cause for fibril formation and the etiology of T2DM. The ultimate goal of this part of the study is to control the growth and nature of amyloid deposits in vivo, which may prove useful for more rational disease management.