MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2

Identification of novel molecular interactions of the mitochondrial fission protein hFis1 : insight into its action mechanism and therapeutic potential

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2009.Mitochondria are dynamic cellular organelles that undergo continuous shape changes within the cell. Two major processes are responsible for the dynamics of mitochondria, namely fission and fusion. A balance of these two opposing events is thought to maintain the normal mitochondrial phenotype in a given cell under normal conditions. Shifting of this balance either way results in abnormal mitochondrial morphologies that are often linked to abnormalities in mitochondrial function and in extreme cases certain disease conditions. Therefore, it has been suggested that mitochondrial dynamics is an important factor in maintaining healthy mitochondria and normal cellular function. The current study investigates the fission process in mammalian mitochondria with specific focus on defining novel molecular interactions of the fission protein hFis1. hFis1 is a mitochondrial outer membrane protein of 17 kDa that acts as a receptor for the cytosolic large GTPase DLP1, which assembles on the outer membrane and constricts the mitochondrial tubules through GTP hydrolysis, bringing about the fission of mitochondria. hFis1 contains six α helices in its N-terminal cytosolic domain, of which the helices 2-5 form tetratricopeptide repeat (TPR)-like folds. It has been shown that the hFis1-DLP1 interaction takes place through this TPR region and is negatively regulated by the N-terminal first α helix of hFis1. The first part of my thesis research focused on studying molecular interactions of hFis1, and its role in the fission process. Earlier studies revealed that mutations in the TPR region of hFis1 resulted in a dominant-negative fission-defective phenotype, suggesting that hFis1 self-interaction occurs between overexpressed mutant hFis1 and the endogenous protein. Using chemical crosslinking and chimera approaches, we found that hFis1 forms homo-oligomeric complexes through self-interaction, in a manner regulated by the first α helix. The specific regions of self-interaction lie within the TPR domain, specifically the linker between the 3rd and 4th α helices, and the Tyrosine 87 residue in the 5th α helix. Disruption of either of these regions through mutations resulted in a drastic reduction of oligomer formation as well as mitochondrial fission. From this study, we conclude that hFis1 forms oligomers through self-interaction in a transient manner as regulated by the N-terminal first α helix of the molecule. We believe that hFis1 oligomerization is a significant event in the fission mechanism that is necessary for the recruitment of cytosolic DLP1 to the outer membrane in order to bring about mitochondrial fission. During the second part of the thesis study, we set out to identify short peptide sequences that bind to hFis1 with the potential for disrupting the molecular interactions required for mitochondrial fission. It has been reported that excessive mitochondrial fission occurs in apoptotic cell death induced by various stimuli, and that blocking fission can rescue cells from programmed cell death in many experimental systems. In order to identify short peptides binding to hFis1 and thus blocking mitochondrial fission and apoptosis, we used random peptide phage display screening. We identified 10 different peptide sequences that bind to the TPR of hFis1. Four of these selected peptides were able to bind to the hFis1 TPR in vitro with Kd values ranging from 9 to 102 μM. Inside mammalian cells, the peptides bring about a reduction of fission resulting in elongated mitochondria. In addition, these peptides were found to drastically reduce staurosporineinduced cytochrome c release from mitochondria in cultured cells, suggesting that they have a therapeutic potential in blocking the apoptotic progression. We believe that these peptides potentially block hFis1 interactions including the hFis-DLP1 interaction as well as the hFis1 self-interaction and, as a result, retard the fission process and, in conditions where excessive fission leads to apoptosis, exert a protective effect. This collective study has investigated novel molecular interactions of the mitochondrial fission protein hFis1 and elucidated their role in controlling mitochondrial morphology. Our results demonstrate that self-interaction of hFis1 is a crucial event for mitochondrial fission and that the hFis1 TPR presents an important binding site to short peptides and other ligands by which mitochondrial fission can be modulated, providing insight that manipulating these interactions can be of potential therapeutic value

Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness

Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes

Late-onset megaconial myopathy in mice lacking group I Paks

Abstract Background Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. Methods We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation. Results dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. Conclusions Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes

RAF/MEK/extracellular signal-related kinase pathway suppresses dendritic cell migration and traps dendritic cells in Langerhans cell histiocytosis lesions

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal-related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)-mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death