The work carried out during the current grant period has identified a new mitochondrial D-loop encoded protein of about 6 kDa, that binds to common sequence motifs on both mitochondrial and nuclear genes. The results of protein purification, Western blot analysis and immunocytochemical analyses demonstrate that the mitochondrial protein is transported to the nucleus as a possible regulatory signal. A PCR based screening of an array of mouse tumors has identified three tumor lines with mutations affecting the sequence of this newly identified protein. Thus, the present proposal is based on the hypothesis that the mitochondrial protein is involved in the regulation of a number of nuclear genes, and it will address some of the long standing questions on the regulation of mitochondrial biogenesis and mechanisms of nuclear-mitochondrial gene interaction, and how these regulatory controls are altered in chemically transformed and established tumor cells as follows: 1) The mouse mitochondrial D-loop encoded protein will be functionally characterized with respect to its DNA binding specificity and its role in transcription stimulation, or down-regulation using in vivo (transient expression in cells) and in vitro transcription systems. Experiments will be carried out to determine if the 6 kDA protein functions as a homodimer or a heterodimer with other nuclear proteins. In the latter case, the protein partner(s) involved in the protein-protein interaction will be characterized. 2) The mode of transport of the D-loop encoded protein to the nuclear compartment and the nature of the transport signal(s) will be studied by in vitro mutagenesis and expression of cDNA constructs in COS cells or human P cells, which lack the mitochondrial DNA. 3) The structure-function relationships and also the possible patho- physiological consequences of the mitochondrial DNA mutations will be determined by analyzing the mutant proteins found in some of the tumor cells and also those derived by in vitro mutagenesis. The effects of in vivo expression of wild type and mutated D-loop proteins in human P cells, and also in cells carrying known mutations on the transcriptional regulation of some of the well characterized nuclear genes will be studied. 4) The nature and types of nuclear genes under the regulatory control of the D-loop encoded mitochondrial protein will be studied by screening the mouse genomic library by in situ protein binding with 32P labeled protein and partial characterization of positive clones by sequence analysis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37CA022762-22
Application #
2825438
Study Section
Special Emphasis Panel (NSS)
Program Officer
Okano, Paul
Project Start
1978-02-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
22
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Chowdhury, A Roy; Long, A; Fuchs, S Y et al. (2017) Mitochondrial stress-induced p53 attenuates HIF-1? activity by physical association and enhanced ubiquitination. Oncogene 36:397-409
Srinivasan, Satish; Guha, Manti; Kashina, Anna et al. (2017) Mitochondrial dysfunction and mitochondrial dynamics-The cancer connection. Biochim Biophys Acta Bioenerg 1858:602-614
Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander et al. (2016) Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene. Ann N Y Acad Sci 1364:52-61
Guha, Manti; Srinivasan, Satish; Guja, Kip et al. (2016) HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression. Cell Discov 2:16045
Dong, Dawei W; Srinivasan, Satish; Guha, Manti et al. (2015) Defects in cytochrome c oxidase expression induce a metabolic shift to glycolysis and carcinogenesis. Genom Data 6:99-107
Guha, M; Srinivasan, S; Ruthel, G et al. (2014) Mitochondrial retrograde signaling induces epithelial-mesenchymal transition and generates breast cancer stem cells. Oncogene 33:5238-50
Bhattacharya, Sabyasachi; Katlinski, Kanstantsin V; Reichert, Maximilian et al. (2014) Triggering ubiquitination of IFNAR1 protects tissues from inflammatory injury. EMBO Mol Med 6:384-97
Guha, Manti; Avadhani, Narayan G (2013) Mitochondrial retrograde signaling at the crossroads of tumor bioenergetics, genetics and epigenetics. Mitochondrion 13:577-91
Tang, Weigang; Chowdhury, Anindya Roy; Guha, Manti et al. (2012) Silencing of IkBýý mRNA causes disruption of mitochondrial retrograde signaling and suppression of tumor growth in vivo. Carcinogenesis 33:1762-8
Srinivasan, Satish; Koenigstein, Alexander; Joseph, Joy et al. (2010) Role of mitochondrial reactive oxygen species in osteoclast differentiation. Ann N Y Acad Sci 1192:245-52

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