Understanding of the mode of action of enzymes involved in the processing of hormonal and polypeptide precursors
In the brain and endocrine glands, the biosynthesis of hormones and biologically active peptides requires the action of highly specialized enzymes in order to modify appropriately biologically inactive precursors into active moieties. These post-translational modifications include amidation, sulfatation, phosphorylation, glycosylation as well as endoproteolytic cleavages in order to yield the myriad of known active hormones. In turn, these molecules can act locally and/or at distant sites where they can be transported. Of paramount importance to this process, the cleavage of the active moiety from the inactive precursor represents a fundamental step in hormone biosynthesis if they are to intervene in cellular communication and in the regulation of the physiologic response of the target organs.
Our comprehension of the hormone excision mechanism from the precursor has increased tremendously following the identification of the intracellular proteolytic enzymes involved in this cleavage, often at pairs of basic amino acids, and collectively known as proprotein convertases. This knowledge was acquired largely through the use of contemporary molecular biology and protein chemistry techniques and allowed the identification of individual members of the convertase family, their gene expression, their mode of action and their localization as well as the unambiguous demonstration of their pivotal role in prohormone processing. Similarly and concomitantly, other enzymes involved in the fashioning of biologically active hormones were also identified and their mode of action deciphered. Furthermore, enzymes and substrates are physically confined within the lumen of specific subcellular organelles, especially secretory granules whereby their composition as well as their integrity ensured a specific “milieu’’ allowing correct production of hormones which will be secreted. This laboratory is focusing his efforts on studying secretory granules present in endocrine cells and in the regulated pathway of secretion that is the one relating the active process of secretion with an extracellular and physiologic stimulus. This intricate mechanism is responsible for the secretion of hormones whose biological role is of tremendous importance, these include corticotropin, endorphin, opiate peptides, insulin glucagon etc. All the biologically active substances are of pivotal importance for growth, homeostasis, survival and reproduction of living organisms.
Experimental approaches
In order to conduct this research successfully, numerous contemporary experimental approaches must be used, these include the following:
Biological relevance of the study
An excellent example of the importance of the above described process is provided by the biosynthesis of the insulin molecule. Indeed, this molecule is first synthesized in the form of proinsulin which is subsequently cleaved by proprotein convertase 1/3 and 2 into insulin, stored into secretory granules and finally released into the circulation following an increase in plasmatic glucose level. This essential mechanism is responsible for maintaining proper circulating glucose level can be severely hampered if not simply compromised in diabetes and this well before actual symptoms of the disease appear. Very often and characteristically, high level of circulating uncleaved or partially cleaved proinsulin can be detected compromising the adequate biological response to raising glucose level. Therefore, this laboratory is aiming to better define the role of proprotein convertase 1/3 as well as to define molecular changes in secretory granules associated with this defect in insulin secretion.
Numerous questions pertaining to changes or impairments associated with diabetes in insulin secretion and, for that matter, potentially other hormone secretion relying on identical secretory mechanism, can be raised.
What is the relationship between this convertase and its immediate cellular or granular surrounding? Is this enzyme interacting in a specific manner with membrane or protein components other than its natural substrates?
Do other molecules in vivo participate in regulating its mode of action and if so are they involved in targeting, transport and/or in modulating enzymatic activitiy?
Is a defect in proinsulin processing responsible for the observed hyperproinsulinemia and if so, what is its molecular basis?
What are the subcellular organelles responsible for the secretion of proinsulin or proinsulin intermediates, are these mature or immature secretory granules?
What is the impact of physiological insults (such as glucotoxicity, lipotoxicity, proteotoxicity, oxidative stress etc.) on the processing of prohormones especially proinsulin into insulin?
In recent years, the incidence of diabetes together with the ensuing losses in quality of life and the economic burden associated with its treatment has taken epidemic proportions especially in the contex of the increasing obesity trend of the population. This laboratory aims at understanding the molecular events underlying the defects in insulin secretion associated with diabetes.
