(DIS)Assembly and Structural Stability of mtHsp60 and its Precursor NaÏve Form

Abstract

Heat shock protein 60kDa is a molecular chaperone (GroEL human homolog) that assists protein folding in mitochondria (mtHsp60). It is synthesized in the cell cytoplasm as a higher molecular weight precursor form (p-mtHsp60) containing a N-terminal targeting sequence, that is cleaved after import into the mitochondrial matrix [1, 2].It has been established, and demonstrated by various techniques, Hsp60 can accumulate in the cytosol, in various pathological conditions (i.e., cancer and chronic inflammatory diseases). The cytosolical Hsp60 accumulation mechanism may occur with or without mitochondrial release concomitantly, so that in the cytosol the two types of 60 kDa chaperonin proteins, (mtHsp60 and its precursor naive form, p-mtHsp60) could coexist [3]. It has been recently observed that in a wide range of concentration, the precursor naive Hsp60 is able to assemble in both heptamers and tetradecamers [4].Key questions still unanswered pertain to the differences in structure-function features that might exist between the well-studied prokaryotic GroEL and the largely unexplored eukaryotic Hsp60 proteins. Moreover, studies on human Hsp60 structure and oligomeric state in vitro could help to validate its role in physiological or pathological cases. In order to pursue this goal, we investigated the (dis)assembly and thermal stability of mtHsp60, p-mtHsp60 and GroEL in vitro, by means of Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC). Complementar Circular Dicroism (CD) measurements were done to follow the change in the secondary structure due to unfolding.[1] Ellis RJ, 2007, Adv Exp Med Biol., 594: 1-13.[2] Jonathan D, 2000, JHC., 48(1): 45-56.[3] D. Chandra, 2007, J. Biol. Chem., 282: 31289-31301.[4] Vilasi S. et al, 2014, Plos One., 9(5): e97657.