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Edited by James Murray, PhD.
Thousands of researchers around the world are studying the connections between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlights how Abcam's range of mitochondrial research tools has contributed to this effort.
Thanks to the endeavors of key mitochondria labs, we now have an improved understanding of the structure and enzymatic mechanism of mitochondrial Complex I, the NADH ubiquinone dehydrogenase.
With upwards of 45 subunits and close to 1 million Da, this is the largest and most complex of the mitochondrial respiratory chain enzymes. However, Complex I is more than just a link in a chain, regulated by substrate availability or subject to mtDNA encoded subunit deficiencies. Regulation of Complex I activity is implicated in the etiology of a wide array of diseases with increasing evidence pointing towards regulation of Complex I by several mechanisms, and by some truly unexpected players.
This month MitoNews focusses on three different pathways regulating Complex I stability and activity, all with different outcomes.
1) Complex I is regulated by transcription factors and cytoplasmic proteins:
The tumor suppressor p53 is a nuclear transcription factor that directly regulates the expression of Bax, a pro-apoptotic member of the Bcl2 family. In work published recently by Kim et al, the authors show that p53 is found in the cytoplasm where it has pro-apoptotic, anti-cancer, properties by indirectly regulating Bax and Complex I activity.
Co -immunoprecipitation experiments show that cytoplasmic p53 binds Bcl-w, liberating Bax from the inactive Bcl-w/Bax heterodimer. Free Bax then migrates to the mitochondrial outer membrane where four critical residues in the C terminus of the Bax protein interact with the ND5 subunit of Complex I located in the inner membrane.
The resulting Bax/Complex I interaction results in down regulation of respiratory chain activity, causing a reduction in reactive oxygen species (ROS) production and cellular invasiveness. Conversely, high levels of Bcl-w or low levels of p53 promote Bcl-w/Bax dimerization and dissociation of the Bax from Complex I which increases Complex I activity, oxidative phosphorylation, ROS and cellular invasiveness.
In this system the anti-apoptotic protein Bcl-xl and pro-apoptotic Bak could substitute for Bcl-w and Bax, respectively, to exert regulation of Complex I suggesting that these family member proteins suppress cell invasion by a common mechanism.
Using a mouse model system, it was observed that xenograft tumors metastasized faster when over-expressing wild type Bcl-w, an effect that was antagonized by over-expression of p53. This suggests that in vivo p53 can also interrupt the Bcl-w/Bax interaction, allowing Bax to inhibit Complex I activity and promote apoptosis.
These studies show that p53 regulates mitochondrial activity and cell invasion by both Bax transcription-dependent (nuclear p53) and Bax transcription- independent (cytoplasmic p53/Bcl-w) mechanisms and that a novel regulatory pathway exists between p53, Bcl-w, Bax and Complex I.
Nuclear and cytoplasmic p53 suppress cell invasion by inhibiting respiratory Complex-I activity via Bcl-2 family proteins. Oncotarget. 2014. Kim E.M., Park J.K., Hwang SG., Kim W.J., Liu Z.G, Kang S.W., and Um H.D.
2) Complex I is regulated by cell surface receptors:
Cell surface receptors, were previously considered unlikely regulators of Complex I activity and the respiratory chain. However, recently Sing et al. published a paper in Cell with evidence to the contrary.
Fat (Ft) cadherins are tumor suppressor genes best characterized in Drosophila, which may be cell adhesion molecules or cell surface receptors and regulate cell proliferation, apoptosis, cell polarity and tissue size via the hippo pathway. In binding studies the authors show that the conserved C terminal domain 2 of the Drosophila plasma membrane Ft binds the N terminal domain of the mitochondrial inner membrane NDUFV2 (a potential regulator of ROS).
For this to occur, the intracellular domain of Ft must be cleaved, by a yet unknown protease, to release a 68 kDa C-terminal cytosolic fragment, termed Ftmito. Using immunofluorescent localization, mitochondrial fractionation and protease protection studies using antibodies against well-established and specific mitochondrial marker proteins, Complex V alpha, porin and NDUFS3, it was observed that this Ftmito fragment is imported into the mitochondrion.
Loss of Ft results in decreased Complex I enzyme assembly and activity, changes in mitochondrial morphology, increased glycolysis and ROS production, subsequently triggering the ROS sensitive JNK pathway. Analysis of blue native PAGE results leads the authors to propose that the 68 kDa Ftmito domain might be yet another Complex I subunit, who’s role is to stabilize the enzyme, upregulate function and permit normal, controlled, proliferation.
In cancer cells lacking Ft, the growth-regulating hippo and PCP pathways become inactive, the mitochondrial Complex I is destabilized, leading to increased ROS production and a Warburg-like aerobic glycolytic metabolic state characterized by normal ATP levels and increased lactate production, a state typical of cancer cells.
This is the first study to show that a cell surface protein functions to directly stabilize the mitochondrial respiratory chain and promote OXPHOS. The authors propose that this regulatory function may be an ancient and conserved role.
The Atypical Cadherin Fat Directly Regulates Mitochondrial Function and Metabolic State. Cell, 2014 Sing A, Tsatskis Y, Fabian L, Hester I, Rosenfeld R, Serricchio M, Yau N, Bietenhader M, Shanbhag R, Jurisicova A, Brill JA, McQuibban GA, McNeill H7.
3) Complex I is regulated by a kinase:
Pogson et al. reported in PLOS Genetics that RNAi knockdown studies of Complex I subunit NDUFA10 (ND42), or its co-chaperone, sicily, in Drosophila specifically induces a mitochondrial hyperfusion phenotype that is indistinguishable from the RNAi knockdown of the PARK6 gene product. PARK6 encodes PTEN-induced kinase 1 (PINK1), a serine/threonine kinase responsible for mitochondrial quality control. Mutations in PARK6 are linked to Parkinson’s disease. Energetic failure of mitochondria to fully import proteins leads to PINK1 accumulation at the mitochondrial outer membrane, which identifies these defective mitochondria for degradation.
The authors validated the connection between PINK1 and ND42 by showing that over-expression of the ND42 subunit (or the chaperone) rescued a PINK1 knockdown which restored mitochondrial morphology, Complex I activity, ATP levels and improved locomotion phenotypes.
The connection between PINK1 and ND42 was also reported by Morais et al. who showed that PINK1 deficient cells have a deficit in phosphorylation of NDUFA10/ND42 at residue Serine 250. This results in down-regulation of Complex I activity, hinting that the kinase, PINK1, may be responsible for phosphorylating this residue.
However, the work of Pogson et al. shows that the overexpression of a mutated, non-phosphorylatable, ND42 variant also rescued the PINK1 knockdown in flies, suggesting that phosphorylation may not be the principle means by which PARK1 regulates Complex I activity.
PARK2, another gene responsible for autosomal recessive Parkinson’s disease, encodes the cytoplasmic enzyme parkin. Parkin is a component of the E3 ubiquitin ligase, recruited by PINK1 on damaged mitochondria, responsible for targeting proteins and organelles for degradation. Over-expression of ND42 did not rescue the knockdown of PARK2 gene product.
The authors propose that PINK1, either directly or indirectly, promotes the assembly of Complex I, enhancing respiratory chain activity. Furthermore, this role in regulating cellular bioenergetics is distinct from the role of PINK1 in mitophagy and mitochondrial quality control.
The complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy. PLoS Genet. 2014. Pogson J.H., Ivatt R.M., Sanchez-Martinez A., Tufi R., Wilson E., Mortiboys H., Whitworth A.J.
PINK1 loss-of-function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science. 2014. Morais VA, Haddad D, Craessaerts K, De Bock PJ, Swerts J, Vilain S, Aerts L, Overbergh L, Grünewald A, Seibler P, Klein C, Gevaert K, Verstreken P, De Strooper B.
As part of our process to consolidate and organize all of our mitochondrial research reagents you may notice that the MitoSciences logo will no longer be displayed on our website. Rest assured, these products will still be available with the same catalogue numbers.