A Quantitative FRET Assay for the Upstream Cleavage Activity of the Integral Membrane Proteases Human ZMPSTE24 and Yeast Ste24

Hsu, Erh Ting; Vervacke, Jeffrey S.; Distefano, Mark D.; Hrycyna, Christine A.

doi:10.1007/978-1-4939-9532-5_21

Cited by 2 publications

(3 citation statements)

References 22 publications

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“…There is little data available on the kinetics of proteolysis of prelamin A based substrates by ZMPSTE24 or comparison of the SY^LL cleavage, which is unique to ZMPSTE24, and the CSIM cleavage, which can also be performed by RCE1. Available studies followed the appearance of the final lamin A product and/or whether it was possible to carboxymethylate the C-terminus [28,35,[37][38][39][40][41][42][43]. Studies involving purified ZMPSTE24 characterized the C^aaX or upstream reaction using a short peptide based on the C-terminal sequence of human K-RAS protein [44] or the yeast a-factor peptide [43] rather than the actual prelamin A sequence.…”

Section: Introductionmentioning

confidence: 99%

“…Available studies followed the appearance of the final lamin A product and/or whether it was possible to carboxymethylate the C-terminus [28,35,[37][38][39][40][41][42][43]. Studies involving purified ZMPSTE24 characterized the C^aaX or upstream reaction using a short peptide based on the C-terminal sequence of human K-RAS protein [44] or the yeast a-factor peptide [43] rather than the actual prelamin A sequence. Another study with purified ZMPSTE24 used in native mass spectrometry to investigate complexes of ZMPSTE24 with peptide substrates and products with the SY^LL site but not the CSIM site [45].…”

Section: Introductionmentioning

confidence: 99%

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A new paradigm for Prelamin A proteolytic processing by ZMPSTE24: the upstream SY^LL cleavage occurs first and there is no CaaX processing by ZMPSTE24

Nie

Spear

Babatz

et al. 2020

Preprint

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Human ZMPSTE24, an integral membrane zinc metalloprotease, is required for conversion of prelamin A to mature lamin A, a component of the nuclear lamina and failure of this processing causes premature ageing disorders. ZMPSTE24 has also been implicated in both type 2 diabetes mellitus and in viral-host response mechanisms, but to date its only confirmed substrate is the precursor for lamin A. Prelamin A is thought to undergo four C-terminal post-translational modifications in the following order: farnesylation, SIM tripeptide cleavage, carboxymethylation and upstream "SY^LL" cleavage. Here we present evidence that the sequence of events does not follow the accepted dogma. We assessed cleavage of long human prelamin A sequence peptides by purified human ZMPSTE24 combined with FRET and mass spectrometry to detect products. Surprisingly, we found that the "SY^LL" cleavage occurs before and independent of the C-terminal CSIM modifications. We also found that ZMPSTE24 does not perform the predicted C^SIM tripeptide cleavage, but rather it removes an IM dipeptide. ZMPSTE24can perform a tripeptide cleavage with a canonical CaaX box (C: cysteine; a: aliphatic; X: any residue), but the C-terminus of prelamin A is not a true CaaX sequence. Regardless of the C-terminal modifications of prelamin A, ZMPSTE24 can perform upstream SY^LL cleavage, thus removing the unwanted farnesylated C-terminus. Therefore, it is failure of SY^LL cleavage, not the C-terminal processing that is the likely cause of progeroid disorders. AbbreviationsDDM, n-dodecyl β-D-maltoside; FRET: fluorescence resonance energy transfer; GFP, green fluorescent protein; HGPS, Hutchinson-Gilford progeria syndrome; MAD-B, mandibuloacral dysplasia type B; RD, restrictive dermopathy; Abz, 2-Aminobenzoyl; Dnp, 2, 4-dinitrophenyl; RFI, relative fluorescence intensity.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A new paradigm for Prelamin A proteolytic processing by ZMPSTE24: the upstream SY^LL cleavage occurs first and there is no CaaX processing by ZMPSTE24

Nie

Spear

Babatz

et al. 2020

Preprint

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“…Because prenylation is not required for substrate recognition, modern mass spectrometry-based methods utilizing standard peptide libraries, such as multiplex substrate profiling by mass spectrometry [67], could be applied to determine Ste24 substrate specificity (this method has been applied successfully to the rhomboid protease [68,69]). Additionally, while multiple researchers (for examples, see [9,13,28,37,70,71]) have performed steady-state kinetic characterization of Ste24, no absolute characterization of turnover and catalytic efficiency has yet been reported. Several technical barriers currently impede development of a standard "platform" for accurate and precise Ste24 steadystate kinetics characterization.…”

Section: Unanswered Questions Unresolved Challengesmentioning

confidence: 99%

Ste24: An Integral Membrane Protein Zinc Metalloprotease with Provocative Structure and Emergent Biology

Goblirsch

Wiener

2020

Journal of Molecular Biology

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Ste24, an integral membrane protein zinc metalloprotease, is found in every kingdom of eukaryotes. It was discovered approximately 20 years ago by yeast genetic screens identifying it as a factor responsible for processing the yeast mating a-factor pheromone. In animals, Ste24 processes prelamin A, a component of the nuclear lamina; mutations in the human ortholog of Ste24 diminish its activity, giving rise to genetic diseases of accelerated aging (progerias). Additionally, lipodystrophy, acquired from the standard highly active antiretroviral therapy used to treat AIDS patients, likely results from off-target interactions of HIV (aspartyl) protease inhibitor drugs with Ste24. Ste24 possesses a novel "α-barrel" structure, consisting of a ring of seven transmembrane α-helices enclosing a large (N 12,000 Å 3 ) interior volume that contains the active-site and substrate-binding region; this "membrane-interior reaction chamber" is unprecedented in integral membrane protein structures. Additionally, the surface of the membrane-interior reaction chamber possesses a strikingly large negative electrostatic surface potential, adding additional "functional mystery." Recent publications implicate Ste24 as a key factor in several endoplasmic reticulum processes, including the unfolded protein response, a cellular stress response of the endoplasmic reticulum, and removal of misfolded proteins from the translocon. Ste24, with its provocative structure, enigmatic mechanism, and recently emergent new biological roles including "translocon unclogger" and (non-enyzmatic) broad-spectrum viral restriction factor, presents far differently than before 2016, when it was viewed as a "CAAX protease" responsible for cleavage of prenylated (farnesylated or geranylgeranylated) substrates. The emphasis of this review is on Ste24 of the "Post-CAAX-Protease Era."

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A Quantitative FRET Assay for the Upstream Cleavage Activity of the Integral Membrane Proteases Human ZMPSTE24 and Yeast Ste24

Cited by 2 publications

References 22 publications

A new paradigm for Prelamin A proteolytic processing by ZMPSTE24: the upstream SY^LL cleavage occurs first and there is no CaaX processing by ZMPSTE24

A new paradigm for Prelamin A proteolytic processing by ZMPSTE24: the upstream SY^LL cleavage occurs first and there is no CaaX processing by ZMPSTE24

Ste24: An Integral Membrane Protein Zinc Metalloprotease with Provocative Structure and Emergent Biology

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