scholarly journals Site specificity determinants for prelamin A cleavage by the zinc metalloprotease ZMPSTE24

2020 ◽  
pp. jbc.RA120.015792
Author(s):  
Timothy D. Babatz ◽  
Eric D. Spear ◽  
Wenxin Xu ◽  
Olivia L. Sun ◽  
Laiyin Nie ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Premature Aging ◽  
Processing Efficiency ◽  
Prelamin A ◽  
Zinc Metalloprotease ◽  
Novel Structure ◽  
Genes Encoding ◽  
Large Water ◽  
Hutchinson Gilford Progeria Syndrome

The integral membrane zinc metalloprotease ZMPSTE24 is important for human health and longevity.  ZMPSTE24 performs a key proteolytic step in maturation of prelamin A, the farnesylated precursor of the nuclear scaffold protein lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that prevent cleavage cause the premature aging disease Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid disorders. ZMPSTE24 has a novel structure, with seven transmembrane spans that form a large water-filled membrane chamber whose catalytic site faces the chamber interior. Prelamin A is the only known mammalian substrate for ZMPSTE24, however, the basis of this specificity remains unclear. To define the sequence requirements for ZMPSTE24 cleavage, we mutagenized the eight residues flanking the prelamin A scissile bond (TRSY↓LLGN) to all other 19 amino acids, creating a library of 152 variants.  We also replaced these eight residues with sequences derived from putative ZMPSTE24 cleavage sites from amphibian, bird, and fish prelamin A.  Cleavage of prelamin A variants was assessed using an in vivo yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency.  We found that residues on the C-terminal side of the cleavage site are most sensitive to changes. Consistent with other zinc metalloproteases, including thermolysin, ZMPSTE24 preferred hydrophobic residues at the P1’ position (Leu647), but in addition, showed a similar, albeit muted, pattern at P2’. Our findings begin to define a consensus sequence for ZMPSTE24 that helps to clarify how this physiologically important protease functions and may ultimately lead to identifying additional substrates.

scholarly journals Cleavage Site Specificity for Processing of Farnesylated Prelamin A by the Zinc Metalloprotease ZMPSTE24

2020 ◽  
Author(s):  
Timothy D. Babatz ◽  
Eric D. Spear ◽  
Wenxin Xu ◽  
Olivia L. Sun ◽  
Laiyin Nie ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Consensus Sequence ◽  
Premature Aging ◽  
Processing Efficiency ◽  
Prelamin A ◽  
Zinc Metalloprotease ◽  
Novel Structure ◽  
Genes Encoding ◽  
Large Water

AbstractThe integral membrane zinc metalloprotease ZMPSTE24 is important for human health and longevity. ZMPSTE24 performs a key proteolytic step in maturation of prelamin A, the precursor of the nuclear scaffold protein lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that prevent cleavage cause the premature aging disease Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid disorders. ZMPSTE24 has a novel structure, with seven transmembrane spans that form a large water-filled membrane chamber whose catalytic site faces the chamber interior. Prelamin A is the only known mammalian substrate for ZMPSTE24, however, the basis of this specificity remains unclear. To define the sequence requirements for ZMPSTE24 cleavage, we mutagenized the eight residues flanking the prelamin A scissile bond (TRSY↓LLGN) to all other 19 amino acids, creating a library of 152 variants. We also replaced these eight residues with sequences derived from putative ZMPSTE24 cleavage sites from amphibian, bird, and fish prelamin A. Cleavage of prelamin A variants was assessed using an in vivo yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. We found that residues on the C-terminal side of the cleavage site are most sensitive to changes. Consistent with other zinc metalloproteases, including thermolysin, ZMPSTE24 preferred hydrophobic residues at the P1’ position (Leu647), but in addition, showed a similar, albeit muted, pattern at P2’. Our findings begin to define a consensus sequence for ZMPSTE24 that helps to clarify how this physiologically important protease functions and may ultimately lead to identifying additional substrates.

scholarly journals Defining substrate requirements for cleavage of farnesylated prelamin A by the integral membrane zinc metalloprotease ZMPSTE24

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0239269
Author(s):  
Kaitlin M. Wood ◽  
Eric D. Spear ◽  
Otto W. Mossberg ◽  
Kamsi O. Odinammadu ◽  
Wenxin Xu ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Mammalian Cells ◽  
Terminal Region ◽  
Proteolytic Processing ◽  
Premature Aging ◽  
Prelamin A ◽  
Zinc Metalloprotease ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome

The integral membrane zinc metalloprotease ZMPSTE24 plays a key role in the proteolytic processing of farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A. Failure of this processing step results in the accumulation of permanently farnesylated forms of prelamin A which cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS), as well as related progeroid disorders, and may also play a role in physiological aging. ZMPSTE24 is an intriguing and unusual protease because its active site is located inside of a closed intramembrane chamber formed by seven transmembrane spans with side portals in the chamber permitting substrate entry. The specific features of prelamin A that make it the sole known substrate for ZMPSTE24 in mammalian cells are not well-defined. At the outset of this work it was known that farnesylation is essential for prelamin A cleavage in vivo and that the C-terminal region of prelamin A (41 amino acids) is sufficient for recognition and processing. Here we investigated additional features of prelamin A that are required for cleavage by ZMPSTE24 using a well-established humanized yeast system. We analyzed the 14-residue C-terminal region of prelamin A that lies between the ZMPSTE24 cleavage site and the farnesylated cysteine, as well 23-residue region N-terminal to the cleavage site, by generating a series of alanine substitutions, alanine additions, and deletions in prelamin A. Surprisingly, we found that there is considerable flexibility in specific requirements for the length and composition of these regions. We discuss how this flexibility can be reconciled with ZMPSTE24’s selectivity for prelamin A.

scholarly journals Defining Substrate Requirements for Cleavage of Farnesylated Prelamin A by the Integral Membrane Zinc Metalloprotease ZMPSTE24

2020 ◽  
Author(s):  
Kaitlin M. Wood ◽  
Eric D. Spear ◽  
Otto W. Mossberg ◽  
Kamsi O. Odinammadu ◽  
Wenxin Xu ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Mammalian Cells ◽  
Terminal Region ◽  
Proteolytic Processing ◽  
Premature Aging ◽  
Prelamin A ◽  
Zinc Metalloprotease ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome

AbstractThe integral membrane zinc metalloprotease ZMPSTE24 plays a key role in the proteolytic processing of farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A. Failure of this processing step results in the accumulation of permanently farnesylated forms of prelamin A which cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS), as well as related progeroid disorders, and may also play a role in physiological aging. ZMPSTE24 is an intriguing and unusual protease because its active site is located inside of a closed intramembrane chamber formed by seven transmembrane spans with side portals in the chamber permitting substrate entry. The specific features of prelamin A that make it the sole known substrate for ZMPSTE24 in mammalian cells are not well-defined. At the outset of this work it was known that farnesylation is essential for prelamin A cleavage in vivo and that the C-terminal region of prelamin A (41 amino acids) is sufficient for recognition and processing. Here we investigated additional features of prelamin A that are required for cleavage by ZMPSTE24 using a well-established humanized yeast system. We analyzed the 14-residue C-terminal region of prelamin A that lies between the ZMPSTE24 cleavage site and the farnesylated cysteine, as well 23-residue region N-terminal to the cleavage site, by generating a series of alanine substitutions, alanine additions, and deletions in prelamin A. Surprisingly, we found that there is considerable flexibility in specific requirements for the length and composition of these regions. We discuss how this flexibility can be reconciled with ZMPSTE24’s selectivity for prelamin A.

scholarly journals ZMPSTE24 Missense Mutations that Cause Progeroid Diseases Decrease Prelamin A Cleavage Activity and/or Protein Stability

2017 ◽  
Author(s):  
Eric D. Spear ◽  
Ehr-Ting Hsu ◽  
Laiyin Nie ◽  
Elisabeth P. Carpenter ◽  
Christine A. Hrycyna ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Stability ◽  
Integral Membrane Protein ◽  
Premature Aging ◽  
Lamin A ◽  
Missense Mutations ◽  
Prelamin A ◽  
Therapeutic Implications ◽  
Zinc Metalloprotease ◽  
Hutchinson Gilford Progeria Syndrome

ABSTRACTThe human zinc metalloprotease ZMPSTE24 is an integral membrane protein critical for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA. After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor, prelamin A, undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) and the related progeroid disorders mandibuloacral dysplasia-type B (MAD-B) and restrictive dermopathy (RD). Here we report a “humanized yeast” system to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability, or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in ER-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib, which may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this “humanized yeast system” can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.

ZMPSTE24, an integral membrane zinc metalloprotease with a connection to progeroid disorders

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Jemima Barrowman ◽  
Susan Michaelis
Keyword(s):  
Human Health ◽  
Scaffold Protein ◽  
Premature Aging ◽  
Mating Pheromone ◽  
Nuclear Scaffold ◽  
Prelamin A ◽  
Zinc Metalloprotease ◽  
Endoproteolytic Cleavage ◽  
Cleavage Step ◽  
Do So

Abstract ZMPSTE24 is an integral membrane zinc metalloprotease originally discovered in yeast as an enzyme (called Ste24p) required for maturation of the mating pheromone a-factor. Surprisingly, ZMPSTE24 has recently emerged as a key protease involved in human progeroid disorders. ZMPSTE24 has only one identified mammalian substrate, the precursor of the nuclear scaffold protein lamin A. ZMPSTE24 performs a critical endoproteolytic cleavage step that removes the hydrophobic farnesyl-modified tail of prelamin A. Failure to do so has drastic consequences for human health and longevity. Here, we discuss the discovery of the yeast and mammalian ZMPSTE24 orthologs and review the unexpected connection between ZMPSTE24 and premature aging.

Emerging candidate treatment strategies for Hutchinson-Gilford progeria syndrome

2017 ◽  
Vol 45 (6) ◽  
pp. 1279-1293 ◽  
Author(s):  
Charlotte Strandgren ◽  
Gwladys Revêchon ◽  
Agustín Sola Carvajal ◽  
Maria Eriksson
Keyword(s):  
De Novo ◽  
Disease Incidence ◽  
Treatment Strategies ◽  
Premature Aging ◽  
Lamin A ◽  
Lmna Gene ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome

Hutchinson-Gilford progeria syndrome (HGPS, progeria) is an extremely rare premature aging disorder affecting children, with a disease incidence of ∼1 in 18 million individuals. HGPS is usually caused by a de novo point mutation in exon 11 of the LMNA gene (c.1824C>T, p.G608G), resulting in the increased usage of a cryptic splice site and production of a truncated unprocessed lamin A protein named progerin. Since the genetic cause for HGPS was published in 2003, numerous potential treatment options have rapidly emerged. Strategies to interfere with the post-translational processing of lamin A, to enhance progerin clearance, or directly target the HGPS mutation to reduce the progerin-producing alternative splicing of the LMNA gene have been developed. Here, we give an up-to-date resume of the contributions made by our and other research groups to the growing list of different candidate treatment strategies that have been tested, both in vitro, in vivo in mouse models for HGPS and in clinical trials in HGPS patients.

scholarly journals Inhibition of DNA damage response at telomeres improves the detrimental phenotypes of Hutchinson–Gilford Progeria Syndrome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Julio Aguado ◽  
Agustin Sola-Carvajal ◽  
Valeria Cancila ◽  
Gwladys Revêchon ◽  
Peh Fern Ong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cellular Senescence ◽  
Genetic Disorder ◽  
Premature Aging ◽  
Telomere Dysfunction ◽  
Damage Response ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome

AbstractHutchinson–Gilford progeria syndrome (HGPS) is a genetic disorder characterized by premature aging features. Cells from HGPS patients express progerin, a truncated form of Lamin A, which perturbs cellular homeostasis leading to nuclear shape alterations, genome instability, heterochromatin loss, telomere dysfunction and premature entry into cellular senescence. Recently, we reported that telomere dysfunction induces the transcription of telomeric non-coding RNAs (tncRNAs) which control the DNA damage response (DDR) at dysfunctional telomeres. Here we show that progerin-induced telomere dysfunction induces the transcription of tncRNAs. Their functional inhibition by sequence-specific telomeric antisense oligonucleotides (tASOs) prevents full DDR activation and premature cellular senescence in various HGPS cell systems, including HGPS patient fibroblasts. We also show in vivo that tASO treatment significantly enhances skin homeostasis and lifespan in a transgenic HGPS mouse model. In summary, our results demonstrate an important role for telomeric DDR activation in HGPS progeroid detrimental phenotypes in vitro and in vivo.

scholarly journals Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613 ◽  
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

scholarly journals Self-assembly of multi-component mitochondrial nucleoids via phase separation

2019 ◽  
Author(s):  
Marina Feric ◽  
Tyler G. Demarest ◽  
Jane Tian ◽  
Deborah L. Croteau ◽  
Vilhelm A. Bohr ◽  
...  
Keyword(s):  
Phase Separation ◽  
Mitochondrial Dysfunction ◽  
Self Assembly ◽  
Size Control ◽  
Premature Aging ◽  
List Type ◽  
Mitochondrial Nucleoids ◽  
Hutchinson Gilford Progeria Syndrome

SummaryMitochondria contain an autonomous and spatially segregated genome. The organizational unit of their genome is the nucleoid, which consists of mitochondrial DNA (mtDNA) and associated architectural proteins. Here, we show that phase separation is the primary physical mechanism for assembly and size-control of the mitochondrial nucleoid. The major mtDNA-binding protein TFAM spontaneously phase separates in vitro via weak, multivalent interactions into viscoelastic droplets with slow internal dynamics. In combination, TFAM and mtDNA form multiphase, gel-like structures in vitro, which recapitulate the in vivo dynamic behavior of mt-nucleoids. Enlarged, phase-separated, yet transcriptionally active, nucleoids are present in mitochondria from patients with the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and are associated with mitochondrial dysfunction. These results point to phase separation as an evolutionarily conserved mechanism of genome organization.HighlightsMitochondrial genomes are organized by phase separation.The main packaging protein TFAM and mtDNA combine to form viscoelastic, multiphase droplets in vitro.Mitochondrial nucleoids exhibit phase behavior in vivo, including dynamic rearrangements and heterogenous organization.Coalescence and enlargement of mt-nucleoids occur upon loss of mitochondrial homeostasis as well as in prematurely aged cells and are associated with mitochondrial dysfunction.

Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

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