DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome.

Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

Regulatory mechanisms of tau protein fibrillation under the conditions of liquid-liquid phase separation.

One of the hallmarks of Alzheimer's disease and several other neurodegenerative disorders is the aggregation of tau protein into fibrillar structures. Building on recent reports that tau readily undergoes liquid-liquid phase separation (LLPS), here we explored the relationship between disease-related mutations, LLPS, and tau fibrillation. Our data demonstrate that, in contrast to previous suggestions, pathogenic mutations within the pseudorepeat region do not affect tau441's propensity to form liquid droplets. LLPS does, however, greatly accelerate formation of fibrillar aggregates, and this effect is especially dramatic for tau441 variants with disease-related mutations. Most important, this study also reveals a previously unrecognized mechanism by which LLPS can regulate the rate of fibrillation in mixtures containing tau isoforms with different aggregation propensities. This regulation results from unique properties of proteins under LLPS conditions, where total concentration of all tau variants in the condensed phase is constant. Therefore, the presence of increasing proportions of the slowly aggregating tau isoform gradually lowers the concentration of the isoform with high aggregation propensity, reducing the rate of its fibrillation. This regulatory mechanism may be of direct relevance to phenotypic variability of tauopathies, as the ratios of fast and slowly aggregating tau isoforms in brain varies substantially in different diseases.

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin.

The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1ß units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(ß)n oligomers (n > 1) while Hp 2-2 occurs in cyclic (α2)n(ß)n oligomers (n > 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp's antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp's capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor's genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.

Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR.

Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs.

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ß-lactam resistance.

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

Purification and identification of native forms of goldfish neuromedin U from brain and gut.

Neuromedin U (NMU) plays important roles in energy homeostasis in rodents and birds. Previously, our group has isolated four cDNAs encoding precursor proteins of NMU from the goldfish brain and gut, and it was assumed that these transcripts are produced by alternative splicing. We have also demonstrated that intracerebroventricular (ICV) injection of putative goldfish NMU inhibits food intake. However, as native goldfish NMU has not yet been identified, we attempted to purify it from goldfish brain and gut extracts. To assess NMU activity in fractions at each purification step, we measured changes in the intracellular concentrations of Ca2+ using HEK293 cells expressing goldfish NMU-R1 or -R2. We isolated a 25-amino-acid peptide (NMU-25) from the brain and gut and found that its primary structure is similar to that of mammalian NMU. Another 21-amino-acid peptide (NMU-21) was purified from the brain, but not from the gut. Furthermore, a 9-amino-acid peptide (NMU-9) identical to the C-terminus of NMU-21 and -25 was also isolated from the brain and gut. Treatment with synthetic NMU-9, -21 and -25 dose-dependently increased the intracellular Ca2+ concentration in mammalian cells expressing goldfish NMU-R1 and -R2. We also examined the effect of ICV-administered synthetic goldfish NMUs on goldfish food intake. NMU-25 inhibited food intake to the same degree as NMU-21. However, the inhibitory effect of NMU-9 was slightly weaker than those of NMU-21 and -25. These results indicate that several molecular forms of NMU exist in the goldfish brain and gut, and that all of them play physiological roles via NMU-R1 and NMU-R2.

Identification and characterization of an alternative cancer-derived PD-L1 splice variant.

Therapeutic blockade of the PD-1/PD-L1 axis is recognized as an effective treatment for numerous cancer types. However, only a subset of patients respond to this treatment, warranting a greater understanding of the biological mechanisms driving immune evasion via PD-1/PD-L1 signaling and other T-cell suppressive pathways. We previously identified a head and neck squamous cell carcinoma with human papillomavirus integration in the PD-L1 locus upstream of the transmembrane domain-encoding region, suggesting expression of a truncated form of PD-L1 (Parfenov et al., Proc Natl Acad Sci USA 111(43):15544-15549, 2014). In this study, we extended this observation by performing a computational analysis of 33 other cancer types as well as human cancer cell lines, and identified additional PD-L1 isoforms with an exon 4 enrichment expressed in 20 cancers and human cancer cell lines. We demonstrate that cancer cell lines with high expression levels of exon 4-enriched PD-L1 generate a secreted form of PD-L1. Further biochemical studies of exon 4-enriched PD-L1 demonstrated that this form is secreted and maintains the capacity to bind PD-1 as well as to serve as a negative regulator on T cell function, as measured by inhibition of IL-2 and IFNg secretion. Overall, we have demonstrated that truncated forms of PD-L1 exist in numerous cancer types, and have validated that truncated PD-L1 can be secreted and negatively regulate T cell function.

Development of a rapid and improved two-dimensional electrophoresis method for the separation of protein lysate.

Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.

The TFF Peptides xP1 and xP4 Appear in Distinctive Forms in the Xenopus laevis Gastric Mucosa: Indications for Different Protective Functions.

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys55 allows monomeric secretion. Furthermore, Cys55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.

Higher Energy Collisional Dissociation Mass Spectrometry of Sulfated O-Linked Oligosaccharides.

Sulfation is the final decoration of mucin-type O-linked oligosaccharides before mucins are released into the lumen of the gastrointestinal, respiratory, and genital tracts. Because only a fraction of oligosaccharides undergo this type of modifications in the Golgi apparatus, sometimes also only by dedicated cells, the glycobiology of these low abundant sulfated oligosaccharides is often overlooked. At the same time, the technology to consistently identify and characterize them has been lagging. We adopted higher energy collisional dissociation to characterize sulfated oligosaccharides from porcine gastric and human salivary MUC5B mucins. With this approach we could generate conclusive spectra up to nonasaccharides. Both singly and doubly sulfated oligosaccharides were characterized. By comparing the fragmentation of low-mass fragments of m/ z 100-320 with standards for six-linked and three-linked sulfate, it could be shown that characteristic fragmentation exists, verifying that porcine gastric mucin contains mostly six-linked sulfate to GlcNAc, whereas human MUC5B contains mostly three-linked Gal. When performing ion-trap MS2 fragmentation, these low-molecular-mass fragments are usually not detected. Hence it can be concluded that to be able to address biological questions of sulfation low-mass fragments are important for the assignment of sulfate position.

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

Natural and targeted isovariants of the rice actin depolymerizing factor 2 can alter its functional and regulatory binding properties.

Actin depolymerizing factors (ADFs) are ubiquitous actin-binding proteins that play essential roles in maintaining cellular actin dynamics by depolymerizing/severing F-actin. Plant ADF isoforms show functional divergence via differential biochemical and cellular properties. We have shown previously that ADF2 of rice (OsADF2) and smooth cordgrass (SaADF2) displayed contrasting biochemical properties and stress response in planta. As a proof-of-concept that amino acid variances contribute to such functional difference, single amino acid mutants of OsADF2 were generated based on its sequence differences with SaADF2. Biochemical studies showed that the single-site amino acid mutations altered actin binding, depolymerizing, and severing properties of OsADF2. Phosphosensitive mutations, such as serine-6>threonine, changed the regulatory phosphorylation efficiency of ADF2 variants. The N-terminal mutations had greater effect on the phosphorylation pattern of OsADF2, whereas C-terminal mutations affected actin binding and severing. The presence of introduced mutations in isovariants of monocot ADF suggests that these residues are significant control points regulating their functional divergence, including abiotic stress response.

Expression and purification of a rapidly degraded protein, TMEM8B-a, in mammalian cell line.

TMEM8B-a protein is the longer, predominant isoform of the TMEM8B gene product, which is a tumor metastasis suppressor in nasopharyngeal carcinoma (NPC) and lung cancer. TMEM8B-a is rapidly degraded via the proteasome pathway mediated by ezrin in many NPC and lung cancer cell lines, but TMEM8B-a is not ubiquitinated. In this study, we report the recombinant production of full-length modified TMEM8B-a in mammalian cells. We used the PiggyBac transposon system to efficiently generate normal and lung cancer cell lines with stable TMEM8B-a protein expression. 293FT cells were the best host cell line to express TMEM8B-a protein. Then, we treated the stable 293FT cell lines with various small-molecule inhibitors and demonstrated that treatment with MG-132 and bortezomib, which target the proteasome and disrupt its function, could prevent TMEM8B-a degradation and induce protein expression in 293FT cells. Finally, we utilized the combination of Twin-Strep-tag and Strep-Tactin XT resin to successfully purify the TMEM8B-a protein. The final yield was estimated to be approximately 10-20 µg of the purified TMEM8B-a per 3.0 × 108 293FT cells.

Sialylated isoforms of apolipoprotein C-III and plasma lipids in subjects with coronary artery disease.

BACKGROUND: Apolipoprotein C-III (ApoC-III), a key regulator of plasma triglyceride (TG), is present in three isoforms, i.e. non-sialylated (ApoC-III0), monosialylated (ApoC-III1) and disialylated (ApoC-III2). We aimed at quantifying the distribution of the ApoC-III glycoforms in patients with angiographically demonstrated coronary artery disease (CAD) according to levels of total ApoC-III plasma concentration. METHODS: ApoC-III glycoforms were quantified by a specifically developed, high-resolution, mass spectrometry method in unrelated CAD patients. Lipoprotein lipase (LPL) activity was estimated by a fluorescence-based method. RESULTS: In 101 statin-treated CAD patients, the absolute concentrations of the three glycoforms similarly increased across ApoC-III quartiles, but the proportion of ApoC-III1 rose whereas that of ApoC-III0 decreased progressively by increasing total ApoC-III concentrations. The proportion of ApoC-III2 was quite constant throughout the whole range of total ApoC-III. A higher proportion of ApoC-III1 reflected an unfavorable lipid profile characterized by high levels of TG, total and low density lipoprotein cholesterol, ApoE and reduced ApoA-I. The correlations between ApoC-III glycoforms and TG were confirmed in 50 statin-free CAD patients. High concentration of total ApoC-III was associated with low LPL activity, while no correlation was found for the relative proportion of glycoforms. CONCLUSIONS: Specific patterns of ApoC-III glycoforms are present across different total ApoC-III concentrations in CAD patients. The inhibitory effect of ApoC-III on LPL appears related to total ApoC-III concentration, but not to the relative proportion of ApoC-III glycoforms.

Characterization of a sandwich ELISA for the quantification of all human periostin isoforms.

BACKGROUND: Periostin (osteoblast-specific factor OSF-2) is a secreted protein occurring in seven known isoforms, and it is involved in a variety of biological processes in osteology, tissue repair, oncology, cardiovascular and respiratory systems or allergic manifestations. To analyze functional aspects of periostin, or the ability of periostin as potential biomarker in physiological and pathological conditions, there is the need for a precise, well-characterized assay that detects periostin in peripheral blood. METHODS: In this study the development of a sandwich ELISA using monoclonal and affinity-purified polyclonal anti-human periostin antibodies was described. Antibodies were characterized by mapping of linear epitopes with microarray technology, and by analyzing cross-reactive binding to human periostin isoforms with western blot. The assay was validated according to ICH/EMEA guidelines. RESULTS: The monoclonal coating antibody binds to a linear epitope conserved between the isoforms. The polyclonal detection antibody recognizes multiple conserved linear epitopes. Therefore, the periostin ELISA detects all known human periostin isoforms. The assay is optimized for human serum and plasma and covers a calibration range between 125 and 4000 pmol/L for isoform 1. Assay characteristics, such as precision (intra-assay: ≤3%, inter-assay: ≤6%), spike-recovery (83%-106%), dilution linearity (95%-126%), as well as sample stability meet the standards of acceptance. Periostin levels of apparently healthy individuals are 864±269 pmol/L (serum) and 817±170 pmol/L (plasma) respectively. CONCLUSION: This ELISA is a reliable and accurate tool for determination of all currently known periostin isoforms in human healthy and diseased samples.

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

BACKGROUND: Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. METHODS: The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. RESULTS: Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. CONCLUSION: The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects.

Quinary structure modulates protein stability in cells.

Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome.

Two glutathione transferase isoforms isolated from juvenile cysts of Taenia crassiceps: identification, purification and characterization.

We identified and characterized the first two glutathione transferases (GSTs) isolated from juvenile cysts of Taenia crassiceps (EC 2.5.1.18). The two glutathione transferases (TcGST1 and TcGST2) were purified in a single-step protocol using glutathione (GSH)-sepharose chromatography in combination with a GSH gradient. The specific activities of TcGST1 and TcGST2 were 26 U mg-1 and 19 U mg-1, respectively, both at 25°C and pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates. The Km(CDNB) and Kcat(CDNB) values for TcGST1 and TcGST2 (0.86 µm and 62 s-1; 1.03 µm and 1.97 s-1, respectively) and Km(GSH) and Kcat(GSH) values for TcGST1 and TcGST2 (0.55 µm and 11.61 s-1; 0.3 µm and 32.3 s-1, respectively) were similar to those reported for mammalian and helminth GSTs. Mass spectrometry analysis showed that eight peptides from each of the two parasite transferases were a match for gi|29825896 glutathione transferase (Taenia solium), confirming that both enzymes are GSTs. The relative molecular masses were 54,000 ± 0.9 for the native enzymes and 27,500 ± 0.5 for the enzyme subunits. Thus, TcGST1 and TcGST2 are dimeric proteins. Optimal TcGST1 and TcGST2 activities were observed at pH 8.5 in the range of 20-55°C and pH 7.5 at 35-40°C, respectively. TcGST1 and TcGST2 were inhibited by cibacron blue (CB), bromosulphophthalein (BST), rose bengal (RB), indomethacin and haematin (Hm) with 50% inhibitory concentrations (IC50) in the µm range. TcGST1 was inhibited in a non-competitive manner by all tested inhibitors with the exception of indomethacin, which was uncompetitive. The discovery of these new GSTs facilitates the potential use of T. crassiceps as a model to investigate multifunctional GSTs.

Structural Characterization of the Allergenic 2S Albumin Cor a 14: Comparing Proteoform Patterns across Hazelnut Cultivars.

The hazelnut allergen Cor a 14 belongs to the 2S albumins, a family of heterodimeric seed storage proteins exhibiting a high degree of structural diversity. Given its relevance as an allergen and the potential to elicit severe reactions, elucidation of the sequence heterogeneity of naturally occurring Cor a 14 is essential for the development of reliable diagnostics and risk evaluation. We therefore performed a comprehensive survey on the proteoforms of Cor a 14 and determined their quantitative distribution in three different hazelnut cultivars by a combinatory HPLC-HRMS approach including bottom-up and intact mass analysis. Compared with the Cor a 14 prototype sequence, we identified three sequence polymorphisms, two of the small and one of the large subunit, and elucidated their specific pairing on the protein level. Furthermore, we located a pronounced microheterogeneity on the protein termini and, for the first time, provide data on varying proteoform patterns between different cultivars of an allergenic seed. Together, these data present the basis for a more detailed investigation on the allergenicity of Cor a 14 in different cultivars and constitute, to be best of our knowledge, the largest set of proteoforms so far reported for a 2S albumin.

Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.