Our objective is to elucidate the molecular basis for structural breakdown in energy deprived cells. Our previous research showed that cell associated glycine is a major factor that maintains cell integrity during depletion of adenosine triphosphate (ATP). ATP depleted cells lose glycine because of compromised transport function. Under these conditions, provision of glycine protects cells from lethal injury. Moreover, glycine, by preserving structure, promotes cell survival. We showed that ATP depleted cells develop porous defects in plasma membranes accompanied by marked aggregation of intramembranous (protein) particles and other structural lesions similar to those induced by complement. These findings suggested that membrane proteins were being redistributed. Plasma membrane lesions were accompanied by specific patterns of cytoplasmic protein aggregation. Glycine prevented not only the functional defects and structural lesions in plasma membranes, but also the cytoplasmic protein alterations. When proteins on the extrinsic surfaces of plasma membranes were selectively cross-linked, the full cytoprotective spectrum of effects mediated by glycine was reproduced, including the promotion of survival. Based on these findings, we postulate that in ATP depleted cells, a single glycine preventable event is initiated at the plasma membrane, triggering not only the formation of porous lesions in membranes, but also a degenerative biochemical cascade that aggregates specific, important proteins and disrupts the cytoplasm. Because equilibrium between phosphorylation and dephosphorylation shifts during ATP depletion, phosphatase activity is likely to be involved in the pathogenesis of these alterations. In view of our findings, we propose the following studies to test our hypotheses regarding the pathogenesis of membrane and cytoplasmic structural damage that is specifically preventable by glycine. 1. Characterization of the molecular basis for cytoplasmic protein aggregation with emphasis on protein dephosphorylation. 2. Definition of the structural basis for increased plasma membrane permeability using freeze-fracture and freeze-etch methods. 3. Comparison of the biochemical and structural bases for cytoprotection conferred by glycine and membrane impermeant cross- linkers. 4. Investigation of membrane protein phosphorylation during ATP depletion. In all these studies, we will focus on aspects of structural and biochemical damage that critically determine loss of viability. Glycine is the most effective agent yet discovered that confers hypoxia tolerance to mammalian cells. Because its actions on cell damage at multiple levels of cytoplasmic organization are so powerful, and since it suppresses biochemical events associated with ATP depletion that are relevant to injury, we are confident that our studies will yield significant new information on hypoxic cell death and its prevention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK037139-13
Application #
2905339
Study Section
Pathology A Study Section (PTHA)
Project Start
1987-07-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
13
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Pathology
Type
Other Domestic Higher Education
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Venkatachalam, Manjeri A; Weinberg, Joel M (2017) Pericytes Preserve Capillary Integrity to Prevent Kidney Hypoxia. J Am Soc Nephrol 28:717-719
Singha, P K; Pandeswara, S; Venkatachalam, M A et al. (2013) Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death. Cell Death Dis 4:e457
Lan, Rongpei; Geng, Hui; Polichnowski, Aaron J et al. (2012) PTEN loss defines a TGF-?-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis. Am J Physiol Renal Physiol 302:F1210-23
Geng, Hui; Lan, Rongpei; Singha, Prajjal K et al. (2012) Lysophosphatidic acid increases proximal tubule cell secretion of profibrotic cytokines PDGF-B and CTGF through LPA2- and G?q-mediated Rho and ?v?6 integrin-dependent activation of TGF-?. Am J Pathol 181:1236-49
Venkatachalam, Manjeri A; Griffin, Karen A; Lan, Rongpei et al. (2010) Acute kidney injury: a springboard for progression in chronic kidney disease. Am J Physiol Renal Physiol 298:F1078-94
Kar, Rekha; Mishra, Nandita; Singha, Prajjal K et al. (2010) Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1. Biochem Biophys Res Commun 399:548-54
Zitta, Karina; Brandt, Berenice; Wuensch, Annegret et al. (2010) Interleukin-1beta regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells. Biochem Biophys Res Commun 399:542-7
Singha, Prajjal K; Yeh, I-Tien; Venkatachalam, Manjeri A et al. (2010) Transforming growth factor-beta (TGF-beta)-inducible gene TMEPAI converts TGF-beta from a tumor suppressor to a tumor promoter in breast cancer. Cancer Res 70:6377-83
Mishra, Nandita; Kar, Rekha; Singha, Prajjal K et al. (2010) Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Delta(12,14)-prostaglandin J2. Biochem Biophys Res Commun 395:17-24
Lan, Rongpei; Geng, Hui; Hwang, Yoon et al. (2010) A novel wounding device suitable for quantitative biochemical analysis of wound healing and regeneration of cultured epithelium. Wound Repair Regen 18:159-67

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