Gopa Biswas

Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells

Alzheimers amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid β (Aβ) peptide associated with the pathogenesis of Alzheimers disease (AD). Here we demonstrate that by virtue of its chimeric NH2-terminal signal, APP is also targeted to mitochondria of cortical neuronal cells and select regions of the brain of a transgenic mouse model for AD. The positively charged residues at 40, 44, and 51 of APP are critical components of the mitochondrial-targeting signal. Chemical cross-linking together with immunoelectron microscopy show that the mitochondrial APP exists in NH2-terminal inside transmembrane orientation and in contact with mitochondrial translocase proteins. Mutational studies show that the acidic domain, which spans sequence 220–290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side. Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism. These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter‐organelle crosstalk

We have investigated the mechanism of mitochondrial–nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m‐chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψm) and electron transport‐coupled ATP synthesis, which was also accompanied by an elevated steady‐state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor‐1 (RyR‐1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+‐responsive factors calineurin, calcineurin‐dependent NFATc (cytosolic counterpart of activated T‐cell‐specific nuclear factor) and c‐Jun N‐terminal kinase (JNK)‐dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF‐κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady‐state [Ca2+]i were normalized in genetically reverted cells which contain near‐normal mtDNA levels. We propose that the mitochondria‐to‐nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion.

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial‐specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic‐free Ca2+ [Ca2+]c. In the present study we show that the mitochondria‐to‐nucleus stress signaling induces invasive phenotypes in otherwise non‐invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor‐specific markers cathepsin L and transforming growth factor β (TGFβ) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4‐ to 6‐fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca2+‐dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with ∼70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.

Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

We have investigated mechanisms of mitochondrial stress-induced phenotypic changes and cell invasion in tumorigenic but poorly invasive human pulmonary carcinoma A549 cells that were partly depleted of mitochondrial DNA (mtDNA). Depletion of mtDNA (genetic stress) caused a markedly lower electron transport-coupled ATP synthesis, loss of mitochondrial membrane potential, elevation of steady state [Ca2+]c, and notably induction of both glycolysis and gluconeogenic pathway enzymes. Markers of tumor invasion, cathepsin L and TGFβ1, were overexpressed; calcium-dependent MAP kinases (ERK1 and ERK2) and calcineurin were activated. The levels of anti-apoptotic proteins Bcl2 and Bcl-XL were increased, and the cellular levels of pro-apoptotic proteins Bid and Bax were reduced. Both mtDNA-depleted cells (genetic stress) and control cells treated with carbonyl cyanide m-chlorophenylhydrazone (metabolic stress) exhibited higher invasive behavior than control cells in a Matrigel basement membrane matrix assay system. MtDNA-depleted cells stably expressing anti-sense cathepsin L RNA, TGFβ1 RNA, or treated with specific inhibitors showed reduced invasion. Reverted cells with 80% of control cell mtDNA exhibited marker protein levels, cell morphology and invasive property closer to control cells. Our results suggest that the mitochondria-to-nucleus signaling pathway operating through increased [Ca2+]c plays an important role in cancer progression and metastasis.

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

Mitochondria to nucleus stress signaling a distinctive mechanism of NFκB/Rel activation through calcineurin-mediated inactivation of IκBβ

Mitochondrial genetic and metabolic stress causes activation of calcineurin (Cn), NFAT, ATF2, and NFκB/Rel factors, which collectively alter the expression of an array of nuclear genes. We demonstrate here that mitochondrial stress–induced activation of NFκB/Rel factors involves inactivation of IκBβ through Cn-mediated dephosphorylation. Phosphorylated IκBβ is a substrate for Cn phosphatase, which was inhibited by FK506 and RII peptide. Chemical cross-linking and coimmunoprecipitation show that NFκB/Rel factor–bound IκBβ forms a ternary complex with Cn under in vitro and in vivo conditions that was sensitive to FK506. Results show that phosphorylation at S313 and S315 from the COOH-terminal PEST domain of IκBβ is critical for binding to Cn. Mutations at S313/S315 of IκBβ abolished Cn binding, inhibited Cn-mediated increase of Rel proteins in the nucleus, and had a dominant-negative effect on the mitochondrial stress–induced expression of RyR1 and cathepsin L genes. Our results show the distinctive nature of mitochondrial stress–induced NFκB/Rel activation, which is independent of IKKα and IKKβ kinases and affects gene target(s) that are different from cytokine and TNFα-induced stress signaling. The results provide new insights into the role of Cn as a critical link between Ca2+ signaling and NFκB/Rel activation.

Bimodal targeting of microsomal CYP2E1 to mitochondria through activation of an N-terminal chimeric signal by cAMP-mediated phosphorylation.

Cytochrome P450 2E1 (CYP2E1) plays an important role in alcohol-induced toxicity and oxidative stress. Recently, we showed that this predominantly microsomal protein is also localized in rat hepatic mitochondria. In this report, we show that the N-terminal 30 amino acids of CYP2E1 contain a chimeric signal for bimodal targeting of the apoprotein to endoplasmic reticulum (ER) and mitochondria. We demonstrate that the cryptic mitochondrial targeting signal at sequence 21–31 of the protein is activated by cAMP-dependent phosphorylation at Ser-129. S129A mutation resulted in lower affinity for binding to cytoplasmic Hsp70, mitochondrial translocases (TOM40 and TIM44) and reduced mitochondrial import. S129A mutation, however, did not affect the extent of binding to the signal recognition particle and association with ER membrane translocator protein Sec61. Addition of saturating levels of signal recognition particle caused only a partial inhibition of CYP2E1 translation under in vitro conditions, and saturating levels of ER resulted only in partial membrane integration. cAMP enhanced the mitochondrial CYP2E1 (referred to as P450MT5) level but did not affect its level in the ER. Our results provide new insights on the mechanism of cAMP-mediated activation of a cryptic mitochondrial targeting signal and regulation of P450MT5 targeting to mitochondria.

Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP‐dependent phosphorylation at Ser128

We have investigated mechanisms of mitochondrial targeting of the phenobarbital‐inducible hepatic mitochondrial P450MT4, which cross‐reacts with antibody to microsomal P4502B1. Results show that P4502B1 and P450MT4 have identical primary sequence but different levels of phosphorylation and secondary structure. We demonstrate that P4502B1 contains a chimeric mitochondrial and endoplasmic reticulum (ER) targeting signal at its N‐terminus. Inducers of cAMP and protein kinase A‐mediated phosphorylation of P4502B1 at Ser128 activate the signal for mitochondrial targeting and modulate its mitochondrial or ER destination. S128A mutation inhibits in vitro mitochondrial transport and also in vivo mitochondrial targeting in COS cells. A fragment of P4502B1 containing the N‐terminal signal and the phosphorylation site could drive the transport of dihydrofolate reductase (DHFR) into mitochondria. Ser128 phosphorylation reduced the affinity of 2B1 protein for binding to SRP, but increased the affinity of the 2B1–DHFR fusion protein for binding to yeast mitochondrial translocase proteins, TOM40 and TIM44, and matrix Hsp70. We describe a novel regulatory mechanism by which cAMP modulates the targeting of a protein to two distinct organelles.

Dioxin-mediated tumor progression through activation of mitochondria-to-nucleus stress signaling

The environmental toxin 2,3,7,8-tetrachlorodibenzodioxin (TCDD) is a known human carcinogen; however, its precise mechanism of action remains unclear. Here we show that TCDD induces mitochondrial dysfunction, stress signaling, and tumor invasion by a mechanism similar to that described for mtDNA-depleted cells. Treatment of C2C12 cells with TCDD disrupted mitochondrial transmembrane potential in a time-dependent fashion and inhibited mitochondrial transcription and translation. TCDD also increased cytosolic [Ca2+]c and RyR1-specific Ca2+ release. These changes were associated with increased calcineurin (CnA) levels and activation of CnA-sensitive NF-κB/Rel (IκBβ-dependent) factors. Cells treated with TCDD displayed resistance to apoptosis, increased expression of the tumor marker cathepsin L, and a high degree of invasiveness as tested by the Matrigel membrane invasion assay. These effects were reversed by the CnA inhibitor FK506, and CnA mRNA silencing suggesting that TCDD triggers a signaling pathway similar to mtDNA depletion. Taken together, these results reveal that TCDD may promote tumor progression in vivo by directly targeting mitochondrial transcription and induction of mitochondrial stress signaling.

Mechanism of mitochondrial stress-induced resistance to apoptosis in mitochondrial DNA-depleted C2C12 myocytes

In this study, we show that partial mitochondrial DNA (mtDNA) depletion (mitochondrial stress) induces resistance to staurosporine (STP)-mediated apoptosis in C2C12 myoblasts. MtDNA-depleted cells show a 3–4-fold increased proapoptotic proteins (Bax, BAD and Bid), markedly increased antiapoptotic Bcl-2, and reduced processing of p21 Bid to active tBid. The protein levels and also the ability to undergo STP-mediated apoptosis were restored in reverted cells containing near-normal mtDNA levels and restored mitochondrial transmembrane potential. Inhibition of apoptosis closely correlated with sequestration of Bax, Bid and BAD in the mitochondrial inner membrane, increased Bcl-2 and Bcl-XL, and inability to process p21 Bid. These factors, together with the reduced activation of caspases 3, 9 and 8 are possible causes of mitochondrial stress-induced resistance to apoptosis. Our results suggest that a highly proliferative and invasive behavior of mtDNA-depleted C2C12 cells is related to their resistance to apoptosis.