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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.

Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential to 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 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-knockout 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.

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 weeks. 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 65-70 weeks, with approximately 40% of male and 75% of female LmnaL648R/L648R mice having near-normal lifespans of 90 weeks (almost 2 years). Starting at ~10 weeks of age, LmnaL648R/L648R mice of both sexes have lower body masses and body fat than controls. By ~20-30 weeks 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 robust model to study the effects of farnesylated prelamin A during physiological aging.

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.

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.

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 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 anin vivoyeast 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.

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. ZMPSTE24 can 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.

Supplemental Digital Content is available in the text., Background: Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models. Methods: We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E–deficient (Apoe–/–) mice with LmnaG609G/G609G mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe–/–LmnaLCS/LCS mice with LysMCre and SM22αCre mice, respectively. Progerin expression was evaluated by polymerase chain reaction and immunofluorescence. Cardiovascular alterations were determined by immunofluorescence and histology in male mice fed normal chow or a high-fat diet. In vivo low-density lipoprotein retention was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein. Cardiac electric defects were evaluated by electrocardiography. Results: Apoe–/–LmnaG609G/G609G mice with ubiquitous progerin expression exhibited a premature aging phenotype that included failure to thrive and shortened survival. In addition, high-fat diet–fed Apoe–/–LmnaG609G/G609G mice developed a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling most aspects of cardiovascular disease observed in patients with HGPS. The same vascular alterations were also observed in Apoe–/–LmnaLCS/LCSSM22αCre mice expressing progerin specifically in VSMCs, but not in Apoe–/–LmnaLCS/LCSLysMCre mice with macrophage-specific progerin expression. Moreover, Apoe–/–LmnaLCS/LCSSM22αCre mice had a shortened lifespan despite the lack of any overt aging phenotype. Aortas of ubiquitously and VSMC-specific progerin-expressing mice exhibited increased retention of fluorescently labeled human low-density lipoprotein, and atheromata in both models showed vulnerable plaque features. Immunohistopathological examination indicated that Apoe–/–LmnaLCS/LCSSM22αCre mice, unlike Apoe–/–LmnaG609G/G609G mice, die of atherosclerosis-related causes. Conclusions: We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.

There are certification and airworthiness requirements relevant to the provision of clean breathing air in the crew and passenger compartments. There have been continuing reports and studies over the years regarding oil fumes in aircraft, including impaired crew performance. Oil fumes are viewed in varying ways ranging from rare seal bearing failures, to low level leakage in normal flight. A Masters of Science (MSc) research degree was undertaken to assess whether there is any gap between the certification requirements for the provision of clean air in crew and passenger compartments, and the theoretical and practical implementation of the requirements using the bleed air system. A comprehensive literature search reviewed applicable certification standards, documented and theoretical understanding of oil leakage. Two types of interviews were conducted to address the research questions. Key aviation regulators were questioned about the process by which they certify and ensure compliance with the clean air requirements. Aerospace engineers and sealing professionals were interviewed about their understanding of how oil may leak past compressor oil bearing seals, and into the air supply under various flight conditions. The outcome of the research showed that there is a gap between the clean air certification requirements, and the theoretical and practical implementation of the requirements using the bleed air system. Low level oil leakage into the aircraft cabin in normal flight operations is a function of the design of the engine lubricating system and bleed air systems, both utilising pressurised air. The use of the bleed air system to supply the regulatory required air quality standards is not being met or being enforced as required.

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.

On board aircraft, the common use of engine compressor, pressurised air to seal the oil bearing chamber and as a source for the cabin bleed-air supply provides a mechanism for low-level oil leakage in routine engine operations. This is of great concern and was discussed previously in this newsletter in a feature entitled ‘Oil bearing seals and aircraft cabin air contamination’ (Sealing Technology April 2016, pages 7–10). Further to this article, Dr Susan Michaelis has now been awarded an MSc for her extensive work in researching the issue of oil leakage past seals in aircraft gas turbine engines. [1] An update on this research and associated initiatives is provided by this article.

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.

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., Summary: The zinc metalloprotease ZMPSTE24 performs the final step of prelamin A processing. Here, a yeast-based system shows differences in protein stability and activity for alleles of ZMPSTE24 that cause progeria disease.

Introduction Since the 1950s pressurised aircraft cabin air has been drawn unfiltered from turbine jet engines as ‘bleed air’. Therefore emissions from engine oil seals leads to contamination of the breathable cabin air. The synthetic oils include a wide range of hazardous substances including the organophosphates, and a complex pyrolysed mixture. Oil seals are reported to produce a noticeable cabin fume event in 1% of flights.1 However, to function oil seals ‘weep’ lubricant at varying stages of normal engine operation,2 so there is always low level air contamination which crew and frequent flyers are cumulatively exposed to for 100 s of hours annually. Reports of adverse effects specifically related to the aircraft environment have continued since the early 1950s.3,4 Short and long-term effects, termed ‘Aerotoxic Syndrome’, primarily involve irritant, sensitising, neurological, neuropsychological, respiratory, cardiovascular and other general effects.4 Methods A review of the various actions undertaken by the aviation manufacturer’s, airlines regulators, governments and independent studies will be outlined. A selection of incident and accident reports and bureau of air safety recommendations will be highlighted. The implications for both aircrew, frequent flyers and susceptible individuals will be discussed. Result The aviation industry has inadequately addressed this long-standing problem, despite it being a clear threat to flight safety, occupational health and safety, occupational and public health. There is a need for further research to be undertaken to help address this important problem relevant to anyone who flies in aircraft as crew or as a passenger. Discussion In view of the need to prevent incidents and accidents related to aircraft contaminated air supplies and the subsequent occupational and public health issues, there is a clear need for action to be taken to assess the scale of the problem and highlight the need for preventative actions.

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.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a well-characterized premature aging disorder caused by mutations in LMNA, the gene encoding the nuclear scaffold proteins lamin A and C. In HGPS and related progerias, processing of prelamin A is blocked at a critical step mediated by the zinc metalloprotease ZMPSTE24. Emerging evidence indicates that LMNA-linked progerias can be grouped into two classes: 1) the processing-deficient, early onset “typical” progerias (e.g. HGPS), and 2) the processing-proficient “atypical” progeria syndromes (APS) that are later in onset. Here we describe a novel progeria syndrome with prominent cutaneous and cardiovascular manifestations belonging to the second class. We suggest the name LMNA-associated cardiocutaneous progeria syndrome (LCPS) for this disorder. Affected patients are normal at birth but undergo progressive cutaneous changes in childhood and die in middle age of cardiovascular complications, including accelerated atherosclerosis, calcific valve disease, and cardiomyopathy. In addition, the proband demonstrated cancer susceptibility, a phenotype rarely described for LMNA-based progeria disorders. The LMNA mutation that causes LCPS is a heterozygous missense mutation resulting in an amino acid substitution (D300G) in the coiled-coil domain of lamin A/C. In skin fibroblasts isolated from the proband, the processing and levels of lamin A and C are normal. However, nuclear morphology is aberrant and rescued by treatment with farnesyltransferase inhibitors (FTIs), as is also the case for HGPS and other laminopathies. Our findings advance knowledge of human LMNA progeria syndromes, and raise the possibility that typical and atypical progerias may converge upon a common mechanism to cause premature aging disease.

Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or nonspecific 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 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., Graphical abstract

Introduction Fumes produced by pyrolysed engine oils have been recognised to contaminate aircraft air supplies since 1953.1,2 The design and use of aircraft air supplies taken from the engine, provides a mechanism for lower level exposure in normal flight. The oils containing a range of hazardous substances including organophosphates, have been associated with ill health amongst aircrew and the travelling public, yet a causal connexion is not recognised. A case controlled study was undertaken to look if health effects could be linked to this unique occupational environment. Methods 275 or 13% of British BAe 146 pilots took part in a case-control study reviewing their workplace environment and health.2 Results 88% of the pilots reported awareness of working in a contaminated air environment, 63% reported symptoms consistent with exposure to oil and other aircraft fluids. 44% reported acute symptoms, with one third reporting longer-lasting adverse effects. 13% displayed a pattern of chronic ill health including a consistent pattern of neurological, neuropsychological, respiratory and other effects, 37–433% above controls. Discussion Despite acute exposure to such substances causing ill health,3 a wide range of evidence and published literature supports the acute and chronic pattern of dysfunction suggesting a causative relationship exists. The illness termed “Aerotoxic Syndrome” is related to a complex but remarkably consistent set of symptoms resulting from this unique occupational environment. The same pattern is seen in aircrew and passengers globally and can be explained by exposure to a variety of chemicals in the jet engine fluids. REFERENCES Treon JF. Industrial Hygiene Quarterly1955;16(3):187–195. Michaelis S. PhD thesis – Health and Flight Safety Implications from Exposure to Contaminated Air in Aircraft, 2010. COT Position paper on Cabin Air, Committee of Toxicity, UK, 2014.

SUMMARY The mating pheromone a -factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a -factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a -factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a -factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a -factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a -factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a -factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila . Thus, additional prenylated signaling molecules resembling a -factor, with as-yet-unknown roles in metazoan biology, may await discovery.

The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical ‘HGPS’) and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.

Summary 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.

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension in addition to its ABC “core” domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor, Tus1p, and is positively regulated by phosphorylation in its ABC core domain at residues Ser-908 and Thr-911. Here we provide evidence that phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of [3H]estradiol-β-17-glucuronide in vitro as compared with wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function, we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 and HAL5, deletion of which increases Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser-251 either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.

The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Deltaycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent-positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.

In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.

Prelamin A undergoes multistep processing to yield lamin A, a structural protein of the nuclear lamina. Prelamin A terminates with a CAAX motif, which triggers farnesylation of a C-terminal cysteine (the C of the CAAX motif), endoproteolytic release of the last three amino acids (the AAX), and methylation of the newly exposed farnesylcysteine residue. In addition, prelamin A is cleaved a second time, releasing 15 more residues from the C terminus (including the farnesylcysteine methyl ester), generating mature lamin A. This second cleavage step is carried out by an endoplasmic reticulum membrane protease, ZMPSTE24. Interest in the posttranslational processing of prelamin A has increased with the recognition that certain progeroid syndromes can be caused by mutations that lead to an accumulation of farnesyl-prelamin A. Recently, we showed that a key cellular phenotype of these progeroid disorders, misshapen cell nuclei, can be ameliorated by inhibitors of protein farnesylation, suggesting a potential strategy for treating these diseases. In this article, we review the posttranslational processing of prelamin A, describe several mouse models for progeroid syndromes, explain the mutations underlying several human progeroid syndromes, and summarize recent data showing that misshapen nuclei can be ameliorated by treating cells with protein farnesyltransferase inhibitors.

The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. After the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 (Face-1) as a suitable candidate to perform one or both of these proteolytic reactions. Evidence has been presented elsewhere establishing that Zmpste24 possesses a zinc-dependent CAAX endopeptidase activity. In the present study, we confirm this CAAX endopeptidase activity with recombinant, membrane-reconstituted Zmpste24 and show that it can accept a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage of prelamin A, we expressed a 33 kDa prelamin A C-terminal tail in insect cells. We demonstrate that this purified substrate possesses a C-terminal farnesylated and carboxyl-methylated cysteine and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. With this substrate, we demonstrate that insect cell membranes bearing recombinant Zmpste24 can also catalyse the second upstream endoproteolytic cleavage.

Numerous proteins, including Ras, contain a C-terminal CAAX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and α-carboxyl methylesterification of the isoprenylated cysteine. This study focuses on the isoprenylcysteine carboxylmethyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes. Ste14p, like all Icmts, has multiple membrane spanning domains, presenting a significant challenge to its purification in an active form. Here, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S. cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary for the carboxylmethylation of isoprenylated substrates. Among the extensive panel of detergents that was screened, optimal solubilization and retention of Ste14p activity occurred with n-dodecyl-β-d-maltoside. The activity of Ste14p could be further optimized upon reconstitution into liposomes. Our expression and purification schemes generate milligram quantities of pure and active Ste14p, which is highly stable under many conditions. Using pure reconstituted Ste14p, we demonstrate quantitatively that Ste14p does not have a preference for the farnesyl or geranylgeranyl moieties in the model substrates N-acetyl-S-farnesyl-l-cysteine (AFC) and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) in vitro. In addition to catalyzing methylation of AFC, we also show that purified Ste14p methylates a known in vivo substrate, Ras2p. Evidence that metals ions are required for activity of Ste14p is also presented. These results pave the way for further characterization of pure Ste14p, as well as determination of its three-dimensional structure.

A general pathway for the internalization of plasma membrane proteins that involves phosphorylation, ubiquitination, recognition and endocytosis has recently emerged from multiple studies in yeast. We refer to this series of events as the PURE pathway. Here we investigate whether the yeast a-factor transporter Ste6p, an ATP-binding cassette protein, utilizes the PURE pathway. Deletion of a 52-amino acid sequence (the ‘A box’) within the linker region of Ste6p has previously been shown to block ubiquitination and endocytosis (Kolling R, Losko S. EMBO J 1997; 16:2251-2261). Using wild-type and mutant forms of GFP-tagged Ste6p, we identified two residues (T613 and S623) within the A box as likely sites of Ste6p phosphorylation important for internalization. Mutation of these residues to alanine blocked ubiquitination and endocytosis of Ste6p, similar to the effect of deleting the entire A box, while substitution with glutamic acid (to mimic phosphorylation) suppressed the ubiquitination and endocytic defects. Importantly, a translational fusion of monoubiquitin to the C-terminus of Ste6p-T613A, S623A or Ste6p-ΔA restored endocytosis, providing strong evidence that the role of phosphorylation is to direct ubiquitination, which in turn is a critical signal for Ste6p internalization. We also identified multiple (five) lysine residues in the linker that are important for Ste6p ubiquitination. Our results demonstrate that Ste6p follows the PURE pathway and that GFP-tagged Ste6p provides a powerful model protein for studies of endocytosis and post-endocytic events in yeast.

Ycf1p is the prototypical member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette (ABC) transporters. Ycf1p resides in the vacuolar membrane and mediates glutathione-dependent transport processes that result in resistance to cadmium and other xenobiotics. A feature common to many MRP proteins that distinguishes them from other ABC transporters is the presence of a hydrophobic N-terminal extension (NTE), whose function is not clearly established. The NTE contains a membrane spanning domain (MSD0) with five transmembrane spans and a cytosolic linker region (L0). The goal of this study was to determine the functional significance of the NTE of Ycf1p by examining the localization and functional properties of Ycf1p partial molecules, expressed either singly or together. We show that MSD0 plays a critical role in the vacuolar membrane trafficking of Ycf1p, whereas L0 is dispensable for localization. On the other hand, L0 is required for transport function, as determined by monitoring cadmium resistance. We also examine an unusual aspect of Ycf1p biology, namely, the posttranslational proteolytic processing that occurs within a lumenal loop of Ycf1p. Processing is shown to be Pep4p dependent and thus serves as a convenient marker for proper vacuolar localization. The processed fragments associate with each other, suggesting that these natural cleavage products contribute together to Ycf1p function.

Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24 -deficient mice ( Zmpste24 −/− ) have indicated a role for Zmpste24 in the processing of CAAX -type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24 −/− mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures—akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24 −/− mice. Zmpste24 −/− mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24 −/− mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.

Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 andRCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif. Ste24p cleaves the carboxyl-terminal “-AAX” from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p. Ste24p also cleaves the amino terminus of a-factor. The mouse genome contains orthologues for both yeast RCE1 and STE24. We previously demonstrated, with a gene-knockout experiment, that mouseRce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins. In this study, we cloned mouseZmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast. Then, to assess the importance ofZmpste24 in development, we generatedZmpste24-deficient mice. Unlike the Rce1knockout mice, Zmpste24-deficient mice survived development and were fertile. Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice. We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAXproteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor. Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility.

Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.