The farnesyl transferase inhibitor (FTI) lonafarnib improves nuclear morphology in ZMPSTE24-deficient fibroblasts from patients with the progeroid disorder MAD-B.

Several related progeroid disorders are caused by defective post-translational processing of prelamin A, the precursor of the nuclear scaffold protein lamin A, encoded by LMNA. Prelamin A undergoes farnesylation and additional modifications at its C-terminus. Subsequently, the farnesylated C-terminal segment is cleaved off by the zinc metalloprotease ZMPSTE24. The premature aging disorder Hutchinson Gilford progeria syndrome (HGPS) and a related progeroid disease, mandibuloacral dysplasia (MAD-B), are caused by mutations in LMNA and ZMPSTE24, respectively, that result in failure to process the lamin A precursor and accumulate permanently farnesylated forms of prelamin A. The farnesyl transferase inhibitor (FTI) lonafarnib is known to correct the aberrant nuclear morphology of HGPS patient cells and improves lifespan in children with HGPS. Importantly, and in contrast to a previous report, we show here that FTI treatment also improves the aberrant nuclear phenotypes in MAD-B patient cells with mutations in ZMPSTE24 (P248L or L425P). As expected, lonafarnib does not correct nuclear defects for cells with lamin A processing-proficient mutations. We also examine prelamin A processing in fibroblasts from two individuals with a prevalent laminopathy mutation LMNA-R644C. Despite the proximity of residue R644 to the prelamin A cleavage site, neither R644C patient cell line shows a prelamin A processing defect, and both have normal nuclear morphology. This work clarifies the prelamin A processing status and role of FTIs in a variety of laminopathy patient cells and supports the FDA-approved indication for the FTI Zokinvy for patients with processing-deficient progeroid laminopathies, but not for patients with processing-proficient laminopathies.

Prelamin A and ZMPSTE24 in premature and physiological aging.

As human longevity increases, understanding the molecular mechanisms that drive aging becomes ever more critical to promote health and prevent age-related disorders. Premature aging disorders or progeroid syndromes can provide critical insights into aspects of physiological aging. A major cause of progeroid syndromes which result from mutations in the genes LMNA and ZMPSTE24 is disruption of the final posttranslational processing step in the production of the nuclear scaffold protein lamin A. LMNA encodes the lamin A precursor, prelamin A and ZMPSTE24 encodes the prelamin A processing enzyme, the zinc metalloprotease ZMPSTE24. Progeroid syndromes resulting from mutations in these genes include the clinically related disorders Hutchinson-Gilford progeria syndrome (HGPS), mandibuloacral dysplasia-type B, and restrictive dermopathy. These diseases have features that overlap with one another and with some aspects of physiological aging, including bone defects resembling osteoporosis and atherosclerosis (the latter primarily in HGPS). The progeroid syndromes have ignited keen interest in the relationship between defective prelamin A processing and its accumulation in normal physiological aging. In this review, we examine the hypothesis that diminished processing of prelamin A by ZMPSTE24 is a driver of physiological aging. We review features a new mouse (LmnaL648R/L648R) that produces solely unprocessed prelamin A and provides an ideal model for examining the effects of its accumulation during aging. We also discuss existing data on the accumulation of prelamin A or its variants in human physiological aging, which call out for further validation and more rigorous experimental approaches to determine if prelamin A contributes to normal aging.

Health consequences of exposure to aircraft contaminated air and fume events: a narrative review and medical protocol for the investigation of exposed aircrew and passengers.

Thermally degraded engine oil and hydraulic fluid fumes contaminating aircraft cabin air conditioning systems have been well documented since the 1950s. Whilst organophosphates have been the main subject of interest, oil and hydraulic fumes in the air supply also contain ultrafine particles, numerous volatile organic hydrocarbons and thermally degraded products. We review the literature on the effects of fume events on aircrew health. Inhalation of these potentially toxic fumes is increasingly recognised to cause acute and long-term neurological, respiratory, cardiological and other symptoms. Cumulative exposure to regular small doses of toxic fumes is potentially damaging to health and may be exacerbated by a single higher-level exposure. Assessment is complex because of the limitations of considering the toxicity of individual substances in complex heated mixtures.There is a need for a systematic and consistent approach to diagnosis and treatment of persons who have been exposed to toxic fumes in aircraft cabins. The medical protocol presented in this paper has been written by internationally recognised experts and presents a consensus approach to the recognition, investigation and management of persons suffering from the toxic effects of inhaling thermally degraded engine oil and other fluids contaminating the air conditioning systems in aircraft, and includes actions and investigations for in-flight, immediately post-flight and late subsequent follow up.

Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control.

Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs. Smt3-K38A/K40A mutant strains also exhibited a defect in the turnover of a soluble PQC model substrate containing the CL1 degron (NES-GFP-Ura3-CL1) localized in the cytoplasm, but not the nucleus. Using human U2OS SUMO1- and SUMO2-KO cell lines, we observed a similar SUMO-dependent pathway for degradation of the mammalian degron-containing PQC reporter protein, GFP-CL1, also only in the cytoplasm but not the nucleus. Moreover, we found that turnover of GFP-CL1 in the cytoplasm was uniquely dependent on SUMO1 but not the SUMO2 paralogue. Additionally, we showed that turnover of GFP-CL1 in the cytoplasm is dependent on the AAA-ATPase, Cdc48/p97. Cellular fractionation studies and analysis of a SUMO1-GFP-CL1 fusion protein revealed that SUMO1 promotes cytoplasmic misfolded protein degradation by maintaining substrate solubility. Collectively, our findings reveal a conserved and previously unrecognized role for SUMO1 in regulating cytoplasmic PQC and provide valuable insights into the roles of sumoylation in PQC-associated diseases.

The Integral Membrane Protein ZMPSTE24 Protects Cells from SARS-CoV-2 Spike-Mediated Pseudovirus Infection and Syncytia Formation.

COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating impact on global public health, emphasizing the importance of understanding innate immune mechanisms and cellular restriction factors that cells can harness to fight viral infections. The multimembrane-spanning zinc metalloprotease ZMPSTE24 is one such restriction factor. ZMPSTE24 has a well-characterized proteolytic role in the maturation of prelamin A, precursor of the nuclear scaffold protein lamin A. An apparently unrelated role for ZMPSTE24 in viral defense involves its interaction with the interferon-inducible membrane proteins (IFITMs), which block virus-host cell fusion by rigidifying cellular membranes and thereby prevent viral infection. ZMPSTE24, like the IFITMs, defends cells against a broad spectrum of enveloped viruses. However, its ability to protect against coronaviruses has never been examined. Here, we show that overexpression of ZMPSTE24 reduces the efficiency of cellular infection by SARS-CoV-2 Spike-pseudotyped lentivirus and that genetic knockout or small interfering RNA-mediated knockdown of endogenous ZMPSTE24 enhances infectivity. We further demonstrate a protective role for ZMPSTE24 in a Spike-ACE2-dependent cell-cell fusion assay. In both assays, a catalytic dead version of ZMPSTE24 is equally as protective as the wild-type protein, indicating that ZMPSTE24's proteolytic activity is not required for defense against SARS-CoV-2. Finally, we demonstrate by plaque assays that Zmpste24-/- mouse cells show enhanced infection by a genuine coronavirus, mouse hepatitis virus (MHV). This study extends the range of viral protection afforded by ZMPSTE24 to include coronaviruses and suggests that targeting ZMPSTE24's mechanism of viral defense could have therapeutic benefit. IMPORTANCE The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has underscored the importance of understanding intrinsic cellular components that can be harnessed as the cell's first line of defense to fight against viral infection. Our paper focuses on one such protein, the integral membrane protease ZMPSTE24, which interacts with interferon-inducible transmembrane proteins (IFITMs). IFITMs interfere with virus entry by inhibiting fusion between viral and host cell membranes, and ZMPSTE24 appears to contribute to this inhibitory activity. ZMPSTE24 has been shown to defend cells against several, but not all, enveloped viruses. In this study, we extend ZMPSTE24's reach to include coronaviruses, by showing that ZMPSTE24 protects cells from SARS-CoV-2 pseudovirus infection, Spike protein-mediated cell-cell fusion, and infection by the mouse coronavirus MHV. This work lays the groundwork for further studies to decipher the mechanistic role of ZMPSTE24 in blocking the entry of SARS-CoV-2 and other viruses into cells.

Abolishing the prelamin A ZMPSTE24 cleavage site leads to progeroid phenotypes with near-normal longevity in mice.

Prelamin A is a farnesylated precursor of lamin A, a nuclear lamina protein. Accumulation of the farnesylated prelamin A variant progerin, with an internal deletion including its processing site, causes Hutchinson-Gilford progeria syndrome. Loss-of-function mutations in ZMPSTE24, which encodes the prelamin A processing enzyme, lead to accumulation of full-length farnesylated prelamin A and cause related progeroid disorders. Some data suggest that prelamin A also accumulates with physiological aging. Zmpste24-/- mice die young, at ∼20 wk. Because ZMPSTE24 has functions in addition to prelamin A processing, we generated a mouse model to examine effects solely due to the presence of permanently farnesylated prelamin A. These mice have an L648R amino acid substitution in prelamin A that blocks ZMPSTE24-catalyzed processing to lamin A. The LmnaL648R/L648R mice express only prelamin and no mature protein. Notably, nearly all survive to 65 to 70 wk, with ∼40% of male and 75% of female LmnaL648R/L648R mice having near-normal lifespans of 90 wk (almost 2 y). Starting at ∼10 wk of age, LmnaL648R/L648R mice of both sexes have lower body masses than controls. By ∼20 to 30 wk of age, they exhibit detectable cranial, mandibular, and dental defects similar to those observed in Zmpste24-/- mice and have decreased vertebral bone density compared to age- and sex-matched controls. Cultured embryonic fibroblasts from LmnaL648R/L648R mice have aberrant nuclear morphology that is reversible by treatment with a protein farnesyltransferase inhibitor. These novel mice provide a model to study the effects of farnesylated prelamin A during physiological aging.

Ultrafine particle levels measured on board short-haul commercial passenger jet aircraft.

BACKGROUND: Airline crew members report adverse health effects during and after inhalation exposure to engine oil fumes sourced to the air supply system onboard commercial and military aircraft. Most investigations into the causal factors of their reported symptoms focus on specific chemical contaminants in the fumes. The adverse health effects reported in aircrew exposed to the aircraft air supply, bled unfiltered off the engine or Auxiliary Power Unit (APU) may be related to particulate exposures, which are widely known to effect health. While oil contaminates the aircraft air supply, some suggest that this will only occur when there is a bearing seal failure, others document that there is low level oil contamination of the air supply during normal engine operation. This brief pilot study explores whether particulate exposure may be associated with the normal engine/APU and air supply operation and to therefore increase the understanding that UFP exposures may have on crew and passengers. METHODS: An ultrafine particle counter was utilised by an experienced airline captain in the passenger cabin of four short-haul commercial passenger aircraft. All flights were under 90 min on aircraft from two different carriers ranging from 7 months to 14 years old. RESULTS: UFP concentrations showed maximum concentrations ranging from 31,300 to 97,800 particles/cm3 when APU was selected on as a source of air on the ground and with engine bleed air and the air conditioning packs selected on during the climb. In 2 of the 4 flights the peaks were associated with an engine oil smell. Increases in UFP particle concentrations occurred with changes in engine/APU power and air supply configuration changes. CONCLUSIONS: This study identified increases in UFP concentrations associated with engine and APU power changes and changes in air supply configuration. These results correlated with times when engine and APU oil seals are known to be less effective, enabling oil leakage to occur. The concentrations reached in the passenger cabins exceeded those taken in other ground-based environments. UFP exposures in aircraft cabins during normal flight indicates there will be health consequences for long serving aircrew and some passengers.

Irritant-induced Asthma Caused by Aerotoxic Syndrome.

PURPOSE: Case series on respiratory features of Aerotoxic Syndrome (AS). The term AS has been coined to describe the spectrum of clinical manifestations after aircraft fume events. Among these manifestations, neurological and respiratory symptoms are the most frequently reported complaints. METHODS: Three cases of AS with relevant respiratory features are presented. RESULTS: Cough and shortness of breath for 6 to12 months were the predominant symptoms in the first two cases. The first case also developed neurological symptoms affecting his central nervous system. In the third case, the patient complained for nine years about an unbearable cough triggered by odors, smells, and a variety of indoor and outdoor irritants, among other symptoms of multiple chemical sensitivity. In all three cases, the respiratory symptoms resolved after appropriate treatment. CONCLUSION: Our report aims at raising awareness on AS and calls for actions to improve the management of patients suffering from this syndrome.

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

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.

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

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.

A humanized yeast system to analyze cleavage of prelamin A by ZMPSTE24.

The nuclear lamins A, B, and C are intermediate filament proteins that form a nuclear scaffold adjacent to the inner nuclear membrane in higher eukaryotes, providing structural support for the nucleus. In the past two decades it has become evident that the final step in the biogenesis of the mature lamin A from its precursor prelamin A by the zinc metalloprotease ZMPSTE24 plays a critical role in human health. Defects in prelamin A processing by ZMPSTE24 result in premature aging disorders including Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid diseases. Additional evidence suggests that defects in prelamin A processing, due to diminished ZMPSTE24 expression or activity, may also drive normal physiological aging. Because of the important connection between prelamin A processing and human aging, there is increasing interest in how ZMPSTE24 specifically recognizes and cleaves its substrate prelamin A, encoded by LMNA. Here, we describe two humanized yeast systems we have recently developed to examine ZMPSTE24 processing of prelamin A. These systems differ from one another slightly. Version 1.0 is optimized to analyze ZMPSTE24 mutations, including disease alleles that may affect the function or stability of the protease. Using this system, we previously showed that some ZMPSTE24 disease alleles that affect stability can be rescued by the proteasome inhibitor bortezomib, which may have therapeutic implications. Version 2.0 is designed to analyze LMNA mutations at or near the ZMPSTE24 processing site to assess whether they permit or impede prelamin A processing. Together these systems offer powerful methodology to study ZMPSTE24 disease alleles and to dissect the specific residues and features of the lamin A tail that are required for recognition and cleavage by the ZMPSTE24 protease.

ZMPSTE24 missense mutations that cause progeroid diseases decrease prelamin A cleavage activity and/or protein stability.

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial 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 the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All mutations 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 endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding 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.

Substrate Insolubility Dictates Hsp104-Dependent Endoplasmic-Reticulum-Associated Degradation.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by ER-associated degradation (ERAD). Although the retrotranslocation of misfolded proteins from the ER has been reconstituted, how a polypeptide is initially selected for ERAD remains poorly defined. To address this question while controlling for the diverse nature of ERAD substrates, we constructed a series of truncations in a single ER-tethered domain. We observed that the truncated proteins exhibited variable degradation rates and discovered a positive correlation between ERAD substrate instability and detergent insolubility, which demonstrates that aggregation-prone species can be selected for ERAD. Further, Hsp104 facilitated degradation of an insoluble species, consistent with the chaperone's disaggregase activity. We also show that retrotranslocation of the ubiquitinated substrate from the ER was inhibited in the absence of Hsp104. Therefore, chaperone-mediated selection frees the ER membrane of potentially toxic, aggregation-prone species.

Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24.

Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or non-specific binding to unintended membrane protein targets. However, distinguishing the binding of drugs to membrane proteins from that of detergents, lipids and cofactors is challenging. Here, we use high-resolution mass spectrometry to study the effects of HIV protease inhibitors on the human zinc metalloprotease ZMPSTE24. This intramembrane protease plays a major role in converting prelamin A to mature lamin A. We monitored the proteolysis of farnesylated prelamin A peptide by ZMPSTE24 and unexpectedly found retention of the C-terminal peptide product with the enzyme. We also resolved binding of zinc, lipids and HIV protease inhibitors and showed that drug binding blocked prelamin A peptide cleavage and conferred stability to ZMPSTE24. Our results not only have relevance for the progeria-like side effects of certain HIV protease inhibitor drugs, but also highlight new approaches for documenting off-target drug binding.

Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast.

Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones.

The Protease Ste24 Clears Clogged Translocons.

Translocation into the endoplasmic reticulum (ER) is the first step in the biogenesis of thousands of eukaryotic endomembrane proteins. Although functional ER translocation has been avidly studied, little is known about the quality control mechanisms that resolve faulty translocational states. One such faulty state is translocon clogging, in which the substrate fails to properly translocate and obstructs the translocon pore. To shed light on the machinery required to resolve clogging, we carried out a systematic screen in Saccharomyces cerevisiae that highlighted a role for the ER metalloprotease Ste24. We could demonstrate that Ste24 approaches the translocon upon clogging, and it interacts with and generates cleavage fragments of the clogged protein. Importantly, these functions are conserved in the human homolog, ZMPSTE24, although disease-associated mutant forms of ZMPSTE24 fail to clear the translocon. These results shed light on a new and critical task of Ste24, which safeguards the essential process of translocation.