Oxaloacetate decarboxylase FAHD1 – a new regulator of mitochondrial function and senescence

Etemad, Solmaz; Petit, Michèle; Weiss, Alexander K. H.; Schrattenholz, André; Baraldo, Giorgia; Jansen‐Dürr, Pidder

doi:10.1016/j.mad.2018.07.007

Cited by 19 publications

(25 citation statements)

References 103 publications

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“…FAHD1 was described previously as regulator of mitochondrial function and senescence [4,5]. According to our current hypothesis, FAHD1 as ODx is involved in the regulation of the TCA cycle flux, probably by regulating the activity of complex II (succinate dehydrogenase, SDH) in the respiratory chain via inhibition by OAA.…”

Section: Discussionmentioning

confidence: 75%

“…Given that mitochondrial dysfunction can induce a spectrum of senescence-like phenotypes [5], and that FAHD1 is involved in the regulation of the TCA cycle flux [6], understanding mFAHD1 in an in vivo model will contribute to the understanding of the underlying physiological or pathological process of FAHD1 deregulation. We have initiated in vivo studies with mFahd1 knockout in a mouse model, that will provide insight into potential phenotypes associated with the absence of mFAHD1, about which we will report in due time.…”

Section: Discussionmentioning

confidence: 99%

“…Whereas many distinct FAH superfamily members were described in prokaryotes [1], the only identified members in eukaryotes are FAH and FAH domain containing proteins 1 and 2 (FAHD1 and FAHD2) [1][2][3]. FAHD1 was identified as oxaloacetate decarboxylase (ODx), and as such acts as a regulatory enzyme in the TCA cycle; accordingly, as it was associated with the regulation of mitochondrial function [4,5].…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

et al. 2020

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FAH domain containing protein 1 (FAHD1) is a mammalian mitochondrial protein, displaying bifunctionality as acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. We report the crystal structure of mouse FAHD1 and structural mapping of the active site of mouse FAHD1. Despite high structural similarity with human FAHD1, a rabbit monoclonal antibody (RabMab) could be produced that is able to recognize mouse FAHD1, but not the human form, whereas a polyclonal antibody recognized both proteins. Epitope mapping in combination with our deposited crystal structures revealed that the epitope overlaps with a reported SIRT3 deacetylation site in mouse FAHD1.culture was grown in 1000 ml NZCYM medium, containing the respective selective antibiotics. At 37 • C the bacteria were amplified to an optical density of 0.4 at 600 nm. Protein expression was induced by the addition of 500 μM isopropyl-1-thio-β-d-galactopyranoside (IPTG) and incubation was continued for 4 h at 37 • C. Bacteria were harvested via centrifugation and stored at −70 • C. Enzyme purificationEnzyme purification of His6/S-double-tagged proteins followed a defined protocol [7]. Recombinant protein was extracted via a three-step purification strategy involving metal affinity chromatography (Ni-NTA), anion exchange chromatography, and gel filtration (SEC). Fractions containing FAHD1 were pooled, concentrated and stored at −70 • C. Gel electrophoresis of two preparations followed by silver staining verified the protein's homogeneity; contaminations were barely visible (<<100 ng/μl).Recombinant mouse FAHD1 (mFAHD1) of concentration in between 1.5 and 2.0 mg/ml (Vivaspin 10 MWCO) was sent for high throughput screening at the HTX laboratory (EMBL, Grenoble). Native mFAHD1 and mFAHD1 supplemented with 1 mM oxalate were screened for crystallization, and hits were obtained in various conditions. A total of 68 crystals were harvested by laser photoablation, cryo-cooled using the CrystalDirect Robot [8,9], and screened for diffraction. Best diffracting crystals of mFAHD1 grew from 25% (w/v) PEG 3350, 0.2 M MgCl 2 and 0.1 M Bis/Tris pH 5.5 and for the oxalate bound protein from 20% (w/v) PEG 4000, 0.05 M MgCl 2 and 0.1 M MES pH 5.5. As the crystals were small needles with volumes between 10 −6 and 10 −5 mm 3 a beam diameter of 15 μm was selected [10]. X-ray diffraction data were collected by the autonomous European Synchrotron Radiation Facility (ESRF) beamline using automatic protocols for the location and optimal centring of crystals [14]. Strategy calculations accounted for flux and crystal volume in the parameter prediction for complete datasets. All data were processed using the automatic pipelines at the ESRF [15]. Statistical analysisKinetic profiles of recombinant protein were obtained with three independent batches of protein preparations (n=3). Data analysis was performed using GraphPad Prism version 5 (www.graphpad.com). Presented data are the mean and standard deviation within 95% confidence interval.

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

et al. 2020

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“…FAHD1, a member of the FAH superfamily of enzymes, has been suggested to play a pivotal role in the regulation of mitochondrial function. Pidder Jansen-Dürr and colleagues discuss the role of FAHD1 as a regulator of mitochondrial function in the context of a potentially reversible form of senescence named by the authors as senescence light (Etemad et al, 2018). On the other hand deregulated activity of SOX2 (Sex-determining region Y box 2), a transcription factor expressed in several fetal and adult tissues, has been linked to age-associated chronic diseases.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Where ageing goes nowadays: Mechanisms, pathways, biomarkers and anti-ageing strategies

Gonos

Chondrogianni

Djordjevic

2019

Mechanisms of Ageing and Development

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Although ageing is (still) an inevitable biological process, humans currently are facing an unprecedented increase in their lifespan. However, healthy ageing has a primate above the simple increase in lifespan and thus the current anti-ageing strategies are switching the focus from suppressing ageing to promote successful ageing and healthspan. The main aim is not just to maximize the length of life, but instead to increase the period of life time spent free of detrimental conditions accompanying old age. Discovering fully the fundamental molecular mechanisms and factors underlying the process of ageing and longevity is a prerequisite for developing appropriate strategies for increasing "healthy lifespan". In this respect ageing research nowadays goes to

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“…Despite their common catalytic center, these enzymes are multi-functional, and most are found in prokaryotes, where they are used to break down compounds retrieved from complex carbon sources 3 . Only three members of this family were identified in eukaryotes so far: the name giving FAH 2 , as well as FAH domain-containing protein 1 (FAHD1) 11 , 12 , 13 , 14 , 15 and FAH domain-containing protein 2 (FAHD2). Depletion of FAHD1 has been associated with impaired mitochondrial respiration 13 , 16 and associated with a reversible type of cellular senescence phenotype 14 that is linked to intermediate potential shortcomings in the electron transport system.…”

Section: Introductionmentioning

confidence: 99%

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Weiss

Holzknecht

Cappuccio

et al. 2019

JoVE

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Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (>98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

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Oxaloacetate decarboxylase FAHD1 – a new regulator of mitochondrial function and senescence

Cited by 19 publications

References 103 publications

Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

Where ageing goes nowadays: Mechanisms, pathways, biomarkers and anti-ageing strategies

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

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