Maiti 2015 PhD Thesis

Maiti P (2016) The role of caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) in regulation of mitochondrial ribosome biogenesis in mammals. PhD Thesis 1-120.

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Maiti P (2016) PhD Thesis

Abstract: CLPP (caseinolytic mitochondrial matrix peptidase proteolytic subunit) is a highly conserved serine protease. Molecular and structural studies in E. coli and other prokaryotes have revealed CLPP specific substrates and the mechanisms underlying their identification and subsequent degradation. These studies showed that ClpXP is involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. Similarly, diverse roles for the eukaryotic CLPP have been suggested. In the filamentous fungus Podospora anserine Clpp depletion promotes longevity. In Caenorhabditis elegans it has been demonstrated that CLPP have a central role in mediating the UPR^mt signals. Loss of function CLPP mutations in humans cause Perrault syndrome that results in ovarian failure and sensorineural hearing loss accompanied with shorter stature. Despite this we still have a very limited knowledge about the functional role of eukaryotic CLPP, its specific substrates and underlying molecular mechanism. In order to decipher the in vivo role of CLPP in mammals we have developed a CLPP deficient mouse model (Clpp^-/-). Interestingly, only about half of Clpp knockout mice according to Mendelian proportion (12,5%) are born from intercrossing of Clpp^+/- mice. These mice are infertile and born ~ 30% smaller than littermates. CLPP deficient mice faithfully replicate the phenotypes observed in human patients. On the molecular level CLPP deficiency leads to an early specific decrease in Complex I activity, followed by a decrease in Complex IV activity later in life. Furthermore, we observed a decrease in mitochondrial translation, which is compensated for by upregulation of mitochondrial transcription. This suggests a direct or indirect role of CLPP in the process of mitochondrial protein synthesis. Gradient sedimentation analysis demonstrates an increase in the steady state levels of small ribosomal subunits, while large ribosomal subunits and monosomes are present in almost normal levels. We also observed an impairment of 12S rRNA assembly into monosomes leading to lower loading of mtmRNAs. This indicates complications in the function of monosomes. Search for CLPXP substrates and interactors revealed two candidates that are likely to be involved in this process. We show that ERAL1 is one of the substrates of CLPP that is likely causing defective 12S rRNA assembly into the small ribosomal subunit. Additionally, p32, a CLPP interactor is permanently bound to the mitoribosomes. We believe that through interaction with CLPXP, these proteins are involved in resolution of stalled ribosomes. We are currently working further on elucidating the molecular mechanism underlying impaired mitochondrial translation.

Labels: MiParea: Respiration 

HRR: Oxygraph-2k 

Labels, 2016-01