AAV9-mediated SMN gene therapy rescues cardiac desmin but not lamin A/C and elastin dysregulation in Smn2B/- spinal muscular atrophy mice.

Structural, functional and molecular cardiac defects have been reported in spinal muscular atrophy (SMA) patients and mouse models. Previous quantitative proteomics analyses demonstrated widespread molecular defects in the severe Taiwanese SMA mouse model. Whether such changes are conserved across different mouse models, including less severe forms of the disease, has yet to be established. Here, using the same high-resolution proteomics approach in the less-severe Smn2B/- SMA mouse model, 277 proteins were found to be differentially abundant at a symptomatic timepoint (post-natal day (P) 18), 50 of which were similarly dysregulated in severe Taiwanese SMA mice. Bioinformatics analysis linked many of the differentially abundant proteins to cardiovascular development and function, with intermediate filaments highlighted as an enriched cellular compartment in both datasets. Lamin A/C was increased in the cardiac tissue, whereas another intermediate filament protein, desmin, was reduced. The extracellular matrix (ECM) protein, elastin, was also robustly decreased in the heart of Smn2B/- mice. AAV9-SMN1-mediated gene therapy rectified low levels of survival motor neuron protein and restored desmin levels in heart tissues of Smn2B/- mice. In contrast, AAV9-SMN1 therapy failed to correct lamin A/C or elastin levels. Intermediate filament proteins and the ECM have key roles in cardiac function and their dysregulation may explain cardiac impairment in SMA, especially since mutations in genes encoding these proteins cause other diseases with cardiac aberration. Cardiac pathology may need to be considered in the long-term care of SMA patients, as it is unclear whether currently available treatments can fully rescue peripheral pathology in SMA.

The Proteome Signatures of Fibroblasts from Patients with Severe, Intermediate and Mild Spinal Muscular Atrophy Show Limited Overlap.

Most research to characterise the molecular consequences of spinal muscular atrophy (SMA) has focused on SMA I. Here, proteomic profiling of skin fibroblasts from severe (SMA I), intermediate (SMA II), and mild (SMA III) patients, alongside age-matched controls, was conducted using SWATH mass spectrometry analysis. Differentially expressed proteomic profiles showed limited overlap across each SMA type, and variability was greatest within SMA II fibroblasts, which was not explained by SMN2 copy number. Despite limited proteomic overlap, enriched canonical pathways common to two of three SMA severities with at least one differentially expressed protein from the third included mTOR signalling, regulation of eIF2 and eIF4 signalling, and protein ubiquitination. Network expression clustering analysis identified protein profiles that may discriminate or correlate with SMA severity. From these clusters, the differential expression of PYGB (SMA I), RAB3B (SMA II), and IMP1 and STAT1 (SMA III) was verified by Western blot. All SMA fibroblasts were transfected with an SMN-enhanced construct, but only RAB3B expression in SMA II fibroblasts demonstrated an SMN-dependent response. The diverse proteomic profiles and pathways identified here pave the way for studies to determine their utility as biomarkers for patient stratification or monitoring treatment efficacy and for the identification of severity-specific treatments.

Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS.

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, glutathione S-transferase pi (GSTP1) exhibited the lowest p-value and highest classification accuracy and Cohen's kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers. SIGNIFICANCE: Oesophageal cancers, including oesophageal adenocarcinoma (OAC) and oesophageal gastric junction cancer (OGJ), are one of the leading causes of cancer mortality worldwide. Curative therapy consists of surgery, either alone or in combination with adjuvant or neoadjuvant chemotherapy or radiation, or combination chemoradiotherapy regimens. There are currently no clinico-pathological means of predicting which patients will benefit from chemotherapeutic treatments. There is therefore an urgent need to improve oesophageal cancer disease management and treatment strategies. This work compared proteomic differences in OAC patients who responded well to chemotherapy as compared to those who did not, using quantitative proteomics prior to treatment commencement. SWATH-MS analysis of plasma (with and without albumin/IgG-depletion) from OAC patients prior to chemotherapy was performed. This approach was adopted to determine whether depletion offered a significant improvement in peptide coverage. Resultant datasets demonstrated that depletion increased peptide coverage significantly. Additionally, there was good quantitative agreement between commonly observed peptides. Data analysis was performed by adopting both univariate as well as multivariate analysis strategies. Differentially abundant proteins were identified between treatment response groups based on tumour regression grade. Such proteins included complement C1q sub-components and GSTP1. This study provides a platform for further work, utilising larger sample sets across different treatment regimens for oesophageal cancer, that will aid the development of 'treatment response prediction assays' for stratification of OAC patients prior to chemotherapy.

Tumour-associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles.

The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here, we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilizing HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T-cell responses in previous studies. Our data thus show the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV.

Lamin A/C dysregulation contributes to cardiac pathology in a mouse model of severe spinal muscular atrophy.

Cardiac pathology is emerging as a prominent systemic feature of spinal muscular atrophy (SMA), but little is known about the underlying molecular pathways. Using quantitative proteomics analysis, we demonstrate widespread molecular defects in heart tissue from the Taiwanese mouse model of severe SMA. We identify increased levels of lamin A/C as a robust molecular phenotype in the heart of SMA mice and show that lamin A/C dysregulation is also apparent in SMA patient fibroblast cells and other tissues from SMA mice. Lamin A/C expression was regulated in vitro by knockdown of the E1 ubiquitination factor ubiquitin-like modifier activating enzyme 1, a key downstream mediator of SMN-dependent disease pathways, converging on ß-catenin signaling. Increased levels of lamin A are known to increase the rigidity of nuclei, inevitably disrupting contractile activity in cardiomyocytes. The increased lamin A/C levels in the hearts of SMA mice therefore provide a likely mechanism explaining morphological and functional cardiac defects, leading to blood pooling. Therapeutic strategies directed at lamin A/C may therefore offer a new approach to target cardiac pathology in SMA.

Quantitative proteomic changes in LPS-activated monocyte-derived dendritic cells: A SWATH-MS study.

Dendritic cells are key immune cells that respond to pathogens and co-ordinate many innate and adaptive immune responses. Quantitative mass spectrometry using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) was performed here to determine the global alterations in monocyte-derived dendritic cells (moDCs) in response to stimulation with lipopolysaccharide (LPS). A moDC library of 4,666 proteins was generated and proteins were quantified at 0, 6 and 24 h post-LPS stimulation using SWATH-MS. At 6 h and 24 h post-LPS exposure, the relative abundance of 227 and 282 proteins was statistically significantly altered (p-value ≤ 0.05), respectively. Functional annotation of proteins exhibiting significant changes in expression between the various time points led to the identification of clusters of proteins implicated in distinct cellular processes including interferon and interleukin signalling, endocytosis, the ER-phagosome pathway and antigen-presentation. In SWATH-MS major histocompatibility complex (MHC) class I proteins were highly upregulated at 24 h, whilst MHC class II proteins exhibited comparatively fewer changes over this period. This study provides new detailed insight into the global proteomic changes that occur in moDCs during antigen processing and presentation and further demonstrates the potential of SWATH-MS for the quantitative study of proteins involved in cellular processes.

Quantitative analysis of hydroxyapatite-binding plasma proteins in genotyped individuals with late-stage age-related macular degeneration.

Age-related macular degeneration (AMD) is associated with the formation of sub-retinal pigment epithelial (RPE) deposits that block circulatory exchange with the retina. The factors that contribute to deposit formation are not well understood. Recently, we identified the presence of spherular hydroxyapatite (HAP) structures within sub-RPE deposits to which several AMD-associated proteins were bound. This suggested that protein binding to HAP represents a potential mechanism for the retention of proteins in the sub-RPE space. Here we performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on plasma samples from 23 patients with late-stage neovascular AMD following HAP-binding. Individuals were genotyped for the high risk CFH variant (T1277C) and binding to HAP was compared between wild type and risk variants. From a library of 242 HAP binding plasma proteins (1% false discovery rate), SWATH-MS revealed significant quantitative differences in the abundance of 32 HAP-binding proteins (p < 0.05) between the two homozygous groups. The concentrations of six proteins (FHR1, FHR3, APOC4, C4A, C4B and PZP) in the HAP eluted fractions and whole plasma were further analysed using ELISA and their presence in sections from human cadaver eyes was examined using immunofluorescence. All six proteins were found to be present in the RPE/choroid interface, and four of these (FHR1, FHR3, APOC4 and PZP) were associated with spherules in sub-RPE space. This study provides qualitative and quantitative information relating to the degree by which plasma proteins may contribute to sub-RPE deposit formation through binding to HAP spherules and how genetic differences might contribute to deposit formation.

Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates.

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.

The major histocompatibility complex class I immunopeptidome of extracellular vesicles.

Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies.

Comparative multiplexed mass spectrometric analyses of endogenously expressed yeast nuclear and cytoplasmic exosomes.

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear-cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the alpha/beta importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.

Hydroquinone dioxygenase from pseudomonas fluorescens ACB: a novel member of the family of nonheme-iron(II)-dependent dioxygenases.

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (K(i) = 14 microM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an alpha2beta2 heterotetramer of 112.4 kDa, which is composed of an alpha-subunit of 17.8 kDa and a beta-subunit of 38.3 kDa. Each beta-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.

The yeast Ski complex is a hetero-tetramer.

The yeast Ski complex assists the exosome in the degradation of mRNA. The Ski complex consists of three components; Ski2, Ski3, and Ski8, believed to be present in a 1:1:1 stoichiometry. Measuring the mass of intact isolated endogenously expressed Ski complexes by native mass spectrometry we unambiguously demonstrate that the Ski complex has a hetero-tetrameric stoichiometry consisting of one copy of Ski2 and Ski3 and two copies of Ski8. To validate the stoichiometry of the Ski complex, we performed tandem mass spectrometry. In these experiments one Ski8 subunit was ejected concomitant with the formation of a Ski2/Ski3/Ski8 fragment, confirming the proposed stoichiometry. To probe the topology of the Ski complex we disrupted the complex and mass analyzed the thus formed subcomplexes, detecting Ski8-Ski8, Ski2-Ski3, Ski8-Ski2, and Ski8-Ski8-Ski2. Combining all data we construct an improved structural model of the Ski complex.

Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry.

We modified and optimized a first generation quadrupole time-of-flight (Q-TOF) 1 to perform tandem mass spectrometry on macromolecular protein complexes. The modified instrument allows isolation and subsequent dissociation of high-mass protein complexes through collisions with argon molecules. The modifications of the Q-TOF 1 include the introduction of (1) a flow-restricting sleeve around the first hexapole ion bridge, (2) a low-frequency ion-selecting quadrupole, (3) a high-pressure hexapole collision cell, (4) high-transmission grids in the multicomponent ion lenses, and (5) a low repetition rate pusher. Using these modifications, we demonstrate the experimental isolation of ions up to 12 800 mass-to-charge units and detection of product ions up to 38 150 Da, enabling the investigation of the gas-phase stability, protein complex topology, and quaternary structure of protein complexes. Some of the data reveal a so-far unprecedented new mechanism in gas-phase dissociation of protein oligomers whereby a tetramer complex dissociates into two dimers. These data add to the current debate whether gas-phase structures of protein complexes do retain some of the structural features of the corresponding species in solution. The presented low-cost modifications on a Q-TOF 1 instrument are of interest to everyone working in the fields of macromolecular mass spectrometry and more generic structural biology.

Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex.

The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task. Here we describe and apply a comprehensive method that combines a mild affinity purification procedure with a multiplexed mass spectrometry approach for the in-depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels. The exosome is an ensemble of primarily 3' --> 5' exoribonucleases and plays a major role in RNA metabolism. The complex has been reported to consist of 11 proteins in molecular mass ranging from 20 to 120 kDa. By using native macromolecular mass spectrometry we measured accurate masses (around 400 kDa) of several (sub)exosome complexes. Combination of these data with proteolytic peptide LC tandem mass spectrometry using a linear ion trap coupled to a FT-ICR mass spectrometer and intact protein LC mass spectrometry provided us with the identity of the different exosome components and (sub)complexes, including the subunit stoichiometry. We hypothesize that the observed complexes provide information about strongly and weakly interacting exosome-associated proteins. In our analysis we also identified for the first time phosphorylation sites in seven different exosome subunits. The phosphorylation site in the Rrp4 subunit is fully conserved in the human homologue of Rrp4, which is the only previously reported phosphorylation site in any of the human exosome proteins. The described multiplexed mass spectrometry-based procedure is generic and thus applicable to many different types of cellular molecular machineries even if they are expressed at endogenous levels.