A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases

López-Pelegrín, Mar; Cerdà-Costa, Núria; Martínez-Jiménez, Francisco; Cintas-Pedrola, Anna; Canals, Albert; Peinado, Juan R.; Martí‐Renom, Marc A.; López-Otı́n, Carlos; Arolas, Joan L.; Gomis‐Rüth, F. Xavier

doi:10.1074/jbc.m113.476580

Cited by 37 publications

(51 citation statements)

References 102 publications

(82 reference statements)

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“…This residue, probably a glutamic acid by analogy with FACE1/Ste24p (PDB entries 2YPT/4IL3; [10,11]), is predicted to be located within a "glutamate helix" [6,18] spanning residues 220-230, which is found after the fourth transmembrane helix as inferred from homology modeling and secondary structure predictions (see Fig. 1B and [7]). Consistently, mutation of the only glutamic acid found within this helix to glutamine (E 222 Q) abolished the complementation activity of HtpX [13].…”

Section: Design Of a Stable Htpx Variantmentioning

confidence: 99%

“…Among them are IM metallopeptidases (MPs) from families M48, M50 and M56 of the MEROPS database (http://merops.sanger.ac.uk), which contain a characteristic metal-binding motif, HEXXH (X stands for any residue; [6][7][8]). These IMMPs participate in a variety of physiological mechanisms that require proteolysis, and they target transcription factors (site-2 proteases), nuclear prelamin A/mating pheromone a-factor (FACE1/Ste24p), and signal molecules that trigger bacterial resistance (BlaR1 and MecR1 from family M56), among others [7]. The biological relevance of IMMPs fosters the need to acquire atomic structural information, mainly by means of X-ray crystallography, in order to be able to modulate the activity of these enzymes.…”

Section: Introductionmentioning

confidence: 99%

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Expression and purification of integral membrane metallopeptidase HtpX

Arolas

García‐Castellanos

Goulas

et al. 2014

Protein Expression and Purification

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The authors state they have no competing financial interest Arolas et al. ! 2! Highlights• The integral membrane metallopeptidase HtpX was overexpressed in E. coli.• A catalytically ablated HtpX mutant was designed to avoid self-cleavage.• The protein was extracted from membranes and purified in octyl-!-D-glucoside.• The protein was purified by cobalt-affinity, anion-exchange and size-exclusion.• The overall system renders milligram amounts of HtpX and fosters structural studies. AbstractLittle is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli.Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His 8 -tag, extracted from the membranes using octyl-!-D-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and sizeexclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members.

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Section: Design Of a Stable Htpx Variantmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression and purification of integral membrane metallopeptidase HtpX

Arolas

García‐Castellanos

Goulas

et al. 2014

Protein Expression and Purification

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“…2A) found in other zinc metallopeptidases (6,7). The histidine residues are most likely Zn(II) ligands, and the glutamate is an invariant catalytic residue involved in the nucleophilic attack of a water molecule on the carbonyl carbon of the scissile peptide bond (6).…”

Section: Oma1 Proteolytic Activity Is Elevated Under Mitochondrial Stmentioning

confidence: 99%

“…Oma1 (overlapping activities with m-AAA 1) is a conserved ATP-independent M48-type zinc metalloprotease, a member of the metazincins family (6,7). Oma1 is intrinsic to the IM, where it exists as a high molecular weight complex (8,9).…”

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confidence: 99%

Stress-triggered Activation of the Metalloprotease Oma1 Involves Its C-terminal Region and Is Important for Mitochondrial Stress Protection in Yeast

Bohovych

Donaldson

Christianson

et al. 2014

Journal of Biological Chemistry

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Background: Oma1 is a conserved membrane-bound protease that forms a high molecular mass complex. Results: Oma1 activity is induced by stress stimuli and required for survival. The activation is linked to changes in Oma1 oligomer stability and involves its C-terminal region. Conclusion: Oma1 function is activated by mitochondrial stress and is important for stress tolerance. Significance: Novel insights into Oma1 function and a potential stress activation mechanism are provided.

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“…Initially postulated to be a backup protease for the m-AAA proteolytic module, Oma1 belongs to the M48C family of conserved metalloproteases 62 with homologues found in both prokaryotes and eukaryotes. 63 Some notable exceptions include Nematoda and Trematoda worms and certain flies (Drosophilidae), which appear to have lost OMA1 homologues throughout their evolution.…”

Section: Oma1 Proteasementioning

confidence: 99%

Metalloproteases of the Inner Mitochondrial Membrane

2017

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The inner mitochondrial membrane (IM) is among the most protein-rich cellular compartments. The metastable IM subproteome where the concentration of proteins is approaching oversaturation creates a challenging protein folding environment with a high probability of protein malfunction or aggregation. Failure to maintain protein homeostasis in such a setting can impair the functional integrity of the mitochondria and drive clinical manifestations. The IM is equipped with a series of highly conserved, proteolytic complexes dedicated to the maintenance of normal protein homeostasis within this mitochondrial subcompartment. Particularly important is a group of membrane-anchored metallopeptidases commonly known as m-AAA and i-AAA proteases, and the ATP-independent Oma1 protease. Herein, we will summarize the current biochemical knowledge of these proteolytic machines and discuss recent advances in our understanding of mechanistic aspects of their functioning.

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A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases

Cited by 37 publications

References 102 publications

Expression and purification of integral membrane metallopeptidase HtpX

Expression and purification of integral membrane metallopeptidase HtpX

Stress-triggered Activation of the Metalloprotease Oma1 Involves Its C-terminal Region and Is Important for Mitochondrial Stress Protection in Yeast

Metalloproteases of the Inner Mitochondrial Membrane

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