GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

Abstract Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular calcium homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. Changes in the actin cytoskeleton also disrupt mitochondria-ER contact sites. This results in lower mitochondrial calcium levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, these findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

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GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

10.1101/2021.03.04.433895 ◽

2021 ◽

Author(s):

Christina Wolf ◽

Alireza Pouya ◽

Sara Bitar ◽

Annika Pfeiffer ◽

Diones Bueno ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Pyruvate Dehydrogenase ◽

Autosomal Recessive ◽

Glutathione Transferase ◽

Pyruvate Dehydrogenase Complex ◽

Underlying Mechanism ◽

Loss Of Function ◽

Mitochondrial Localization ◽

Cellular Ca ◽

Dehydrogenase Complex

AbstractCharcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 in a redoxdependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. Changes in the actin cytoskeleton also disrupt mitochondria-ER contact sites. This results in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, these findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

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The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

10.1101/2020.06.02.130369 ◽

2020 ◽

Author(s):

Jaehyoun Lee ◽

Seunghee Oh ◽

Saikat Bhattacharya ◽

Ying Zhang ◽

Laurence Florens ◽

...

Keyword(s):

Ionic Strength ◽

Pyruvate Dehydrogenase ◽

Biochemical Analysis ◽

Pyruvate Dehydrogenase Complex ◽

Size Exclusion ◽

Dependent Manner ◽

Acetyl Coa ◽

Cell Extracts ◽

Exclusion Chromatography ◽

Dehydrogenase Complex

ABSTRACTThe pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

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The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

PLoS ONE ◽

10.1371/journal.pone.0243489 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243489

Author(s):

Jaehyoun Lee ◽

Seunghee Oh ◽

Saikat Bhattacharya ◽

Ying Zhang ◽

Laurence Florens ◽

...

Keyword(s):

Ionic Strength ◽

Pyruvate Dehydrogenase ◽

Biochemical Analysis ◽

Pyruvate Dehydrogenase Complex ◽

Size Exclusion ◽

Dependent Manner ◽

Acetyl Coa ◽

Cell Extracts ◽

Exclusion Chromatography ◽

Dehydrogenase Complex

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

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Inhibition of the Plastidic Pyruvate Dehydrogenase Complex in Isolated Plastids of Oat

Zeitschrift für Naturforschung C ◽

10.1515/znc-1994-7-806 ◽

1994 ◽

Vol 49 (7-8) ◽

pp. 421-426 ◽

Cited By ~ 5

Author(s):

Andrea Golz ◽

Hartmut K. Lichtenthaler

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Pyruvate Dehydrogenase ◽

Fatty Acid Biosynthesis ◽

De Novo ◽

Pyruvate Dehydrogenase Complex ◽

Dependent Manner ◽

Acid Biosynthesis ◽

Acetyl Coa ◽

Dehydrogenase Complex

The activity of the plastidic pyruvate dehydrogenase complex (pPDHC) is one source of acetyl-CoA in plastids of higher plants needed for de novo fatty acid biosynthesis. This plastidic enzyme reaction is specifically inhibited by acetylmethylphosphinate (AMPI), a com pound which had hitherto been known only as an inhibitor of the mitochondrial pyruvate dehydrogenase complex (mPDHC). In the test system of isolated intact oat plastids (Avena sativa) [2-14C]pyruvate was used for de novo fatty acid biosynthesis. The incorporation of label from [2-14C]pyruvate in fatty acids was inhibited by AMPI in a dose-dependent manner. The inhibition rose with increasing preincubation time of plastids with the inhibitor. I50 values for the inhibition of de novo fatty acid biosynthesis from [2-14C]pyruvate by AMPI for isolated etioplasts and chloroplasts were 4.5 and 80 μm , respectively. The activity of the pPDHC decreased during greening of oat seedlings, as is seen from the decreasing incorporation of [2-14C]pyruvate into fatty acids during the light-induced transformation of etioplasts into chloroplasts. In contrast to the decreasing pPDHC activity, the activity of the plastidic acetyl-C oA synthetase (ACS), which transfers acetate to acetyl-CoA, rose parallel to the transformation of etioplasts into chloroplasts. During the assay time of 20 min we could not detect an incorporation of radiolabel from pyruvate or acetate into β-carotene or any other carotenoid

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Selective proteolysis of the protein X subunit of the bovine heart pyruvate dehydrogenase complex. Effects on dihydrolipoamide dehydrogenase (E3) affinity and enzymic properties of the complex

Biochemical Journal ◽

10.1042/bj2780423 ◽

1991 ◽

Vol 278 (2) ◽

pp. 423-427 ◽

Cited By ~ 22

Author(s):

J C Neagle ◽

J G Lindsay

Keyword(s):

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Catalytic Efficiency ◽

Intact Protein ◽

Loss Of Function ◽

Complex Activity ◽

Bovine Heart ◽

Protein X ◽

Dehydrogenase Complex ◽

Selective Proteolysis

Selective proteolysis of the protein X subunit of native bovine heart pyruvate dehydrogenase complex may be accomplished without loss of overall complex activity. Partial loss of function occurs if Mg2+ and thiamin pyrophosphate are not present during proteinase arg C treatment as these cofactors are necessary to prevent cleavage of the E1 alpha subunit. Specific degradation of component X leads to marked alterations in the general enzymic properties of the complex. Lipoamide dehydrogenase (E3) exhibits a decreased affinity for the core assembly and the complex is much more susceptible to inactivation at high ionic strength. The inactive form of the complex is not readily re-activated by removal of salt. It appears that intact protein X and specifically the presence of its cleaved lipoyl domain is not essential for maintenance of an enzymically active pyruvate dehydrogenase complex. However, this protein has an important structural role in promoting the correct association of E3 with the E2 core assembly, an interaction that is required for optimal catalytic efficiency of the complex.

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