Mixture Of Isoforms Research Articles

Mutant p53 Drives the Development of Myelodysplastic Syndromes Via Dysregulating Pre-mRNA Splicing in Hematopoietic Stem and Progenitor Cells

Despite the clinical importance of TP53 mutations, to date there have been no studies on the functional impact of TP53 mutations on the initiation and progression of MDS. We found that heterozygous p53 mutant mice ( p53R248W/+) show extended survival and died within 15-20 months after birth. p53 mutant mice had hypercellular bone marrow and displayed cytopenia, thrombocytopenia, and anemia. Approximately 60% of p53R248W/+ mice developed MDS based upon pathological analysis of bone marrow (BM) and peripheral blood smears, while the remaining p53R248W/+ mice developed lymphoma and sarcoma. We further showed that mutant p53-driven MDS are transplantable. Thus, we have generated a mouse model of MDS that has some features of human MDS with TP53 mutations. To understand how mutant p53 drives MDS development, we performed RNA-seq studies to compare gene expression in hematopoietic stem and progenitor cells (HSPCs). GSEA analysis revealed that spliceosome genes were significantly downregulated in middle-aged p53 R248W/+ HSPCs compared to age-matched p53 +/+ HSPCs. We confirmed that the expression of splicing factors, such as Prpf3 and Prpf4, was significantly downregulated in middle-aged p53 R248W/+ HSPCs. We conducted alternative splicing analysis utilizing MISO (Mixture of Isoforms) algorithm to identify differences in exon inclusion ratio between WT and mutant HSPCs. We observed differential splicing of all classes of alternative splicing events, including Cassette Exons (CE), Competing 5′ and 3′ splice sites (A5' or A3'SS), Mutually Exclusive Exons (MXE), and Retained Introns (RI) in p53 R248W/+ cells compared to that of the WT HSPCs. Notably, we found that murine p53 mutant HSPCs exhibited aberrant splicing of key regulators of NFκBactivation, such as USP15. USP15 overexpression promotes NFκB expression through inhibiting its ubiquitination, whereas NFκB promotes USP15 expression. p53 mutant HSPCs tend to skip exon 7 and express high levels of short isoform of Usp15. The short isoform of Usp15, but not the long isoform of Usp15, activates NFκB in HSPCs, manifested by increased phosphorylation of p65. To determine the impact of mutant p53 on pre-mRNA splicing in human HSPCs, we ectopically expressed GFP or p53 R248W in human cord blood CD34 + cells and performed RNA-seq in transduced cells (GFP +). Human HSPCs expressing mutant p53 displayed alterations in pre-mRNA splicing compared to HSPCs expressing GFP. We found that human HSPCs expressing mutant p53 display aberrant splicing in key regulator of NFκB such as IKBKE (Inhibitor of nuclear factor kappa-B kinase subunit epsilon). Human HSPCs expressing mutant p53 tend to include exon 7 and express high levels of long isoform of IKBKE. The long isoform of IKBKE, but not the short isoform of IKBKE, activates NFκB in human HSPCs. Many proinflammatory cytokine genes are NFκB targets and we observed increased levels of pro-inflammatory cytokines, including IL-1β and IL-6, in the BM of middle-aged p53 mutant mice. Notably, we observed increased levels of IL-1β and IL-6 in the BM of MDS or AML patients with TP53 mutations. TP53 mutations co-occur with splicing factor mutations in MDS. To determine whether TP53 and SRSF2 mutations cooperate in pathogenesis of MDS, we have generated p53 R248W/+SRSF2 P95H/+ mice. We found that the survival of recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells was significantly decreased compared to recipients repopulated with WT, p53 R248W/+, or SRSF2 P95H/+ BM cells. All recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells died within 6 months and developed MDS or leukemia. Thus, we demonstrate that TP53 and SRSF2 mutations cooperate in MDS or leukemia development. In summary, we discovered that mutant p53 dysregulates pre-mRNA splicing in key regulators of inflammatory response during aging, thereby generating a chronic inflammatory microenvironment to drive MDS pathogenesis.

O-067 Does the measurement of different AMH isoforms in poor responder patients improve the prediction of ovarian response?

Abstract Study question Does the measurement of AMH isoforms in low ovarian reserve patients improve the prediction of the number of oocytes after ovarian stimulation (OS)? Summary answer AMH assays measuring a broader range of AMH isoforms combined with antral follicle count (AFC) improve the prediction of oocytes collected after OS. What is known already Granulosa cells secrete AMH as a non-active prohormone (proAMH). After cleavage, it forms a complex (AMHN,C) which activates the receptor (AMHR2). Circulating AMH is a mixture of isoforms (proAMH, AMHN,C and other sub-fragments) after proteolysis, targeted by the monoclonal antibodies included in the AMH assays commonly used in clinical practice, potentially affecting the quantification of the hormone. Novel AMH assays include antibodies directed towards a broader range of the molecule. They may provide more accurate and reliable results, especially important in poor responders, as results may involve clinical decisions on whether or not to proceed with OS. Study design, size, duration Prospective observational study measuring AMH isoforms using five different AMH assays, including 72 women with low ovarian reserve performing OS for IVF/ICSI at a tertiary referral fertility center, from February 2019 to December 2021. Low ovarian reserve was defined as AMH serum levels <1.1ng/ml (Elecsys, Roche), following Bologna criteria. All patients performed OS for IVF/ICSI with antagonist protocol. Participants/materials, setting, methods On day 2/3 of cycle, prior to initiating the OS, AFC, serum FSH, LH, estradiol, progesterone and AMH levels were measured using Elecsys assay (Roche). Extra serum samples were frozen at -20C for subsequent analysis, using four different AMH assays (AnshLabs, Texas): AL-196 (PCOCheck ELISA), AL-124 (PicoAMH ELISA), AL-105 (US-AMH ELISA) and AL-133 (Total Mature-AMH ELISA). Ethical approval was obtained (Research Ethics Committee-REFA033c) and informed consent was signed by all participants. Main results and the role of chance Patient characteristics were [Median(IQR)]: age:39(36-42)years, BMI:28.51(26.1-30.1) Kg/m2, AFC:5.5(4-7), FSH:8.23(6.4-12.5) mIU/mL, LH 6.54(4.51-8.91) mIU/mL, Estradiol: 41.18(28.47-56.44)pg/mL, AMH-Elecsys: 0.64(0.29-0.81)ng/mL, AL-196:0.64(0.39-0.99)ng/mL, AL-124:0.79(0.49-1.24ng/mL, AL-105: 0.89(0.57-1.39)ng/mL, AL-133:1.06(0.63-1.59)ng/mL. Stimulation outcomes were: follicle number on the day of trigger (Fdot):5(3-7), cumulus-oocyte-complexes (COC’s):3(2-4.5) and metaphase II oocytes (MII):3(1-4). Spearman correlation was performed between AMH assays, AFC and OS outcomes. Elecsys revealed a good correlation with the other AMH assays (rs = 0.60-0.63,p<0.001), however, it was higher among ELISA assays (rs = 0.954-0.993,p<0.001). All AMH assays showed a significant positive correlation with AFC, Fdot, COC’s and MII. However, AL-196 showed the highest correlation (rs = 0.524, rs = 0.615, rs = 0.594, rs = 0.595, respectively;p<0.001). Different models combining one AMH assay plus AFC were created to predict COC’s and MII: AFC+AL-196 revealed the best prediction (Adjusted R2=0.474[p < 0.001] and 0.485[p < 0.001], respectively) when compared to the same AMH assay alone. AUROC analysis was performed to investigate which model predicted better < =3COC’s after OS. Although all models demonstrated fair values, AUROC was better for AFC+AL-196 model (AUROC:0.796), yet no significant differences were seen when compared to AUROC for AFC+AMH-Elecsys (p = 0.39). AFC and AMH were good predictors for COC’s collected [RR(95%CI):1.49(1.11-2.04),p<0.01; RR(95%CI):1.26(1.13-1.49),p<0.001, respectively). AFC+AMH-Elecsys predicted COC’s better than AFC alone (R2=0.47 vs 0.29, respectively;Performance-Score:42.56% vs 13.21%, respectively). AFC+AL-196 had the highest performance (R2=0.599;Performance-Score:90.12%). Limitations, reasons for caution Although clinical assessment was performed in the same centre following the same methodology, inter-observer variability for AFC is a limitation. Besides, fresh AMH serum samples were analysed using Elecsys assay, whereas frozen serum samples were shipped to Ansh Lab (Texas) for batched analysis. Wider implications of the findings For patients with serum AMH<1.1ng/mL, a wide range of oocytes might be expected after OS. Novel AMH assays, targeting a wider broad of the molecule, together with AFC, can be used for these patients to obtain a better prediction for clinical outcomes and anticipate very poor response. Trial registration number Not applicable

Cross-seeding of WT amyloid-β with Arctic but not Italian familial mutants accelerates fibril formation in Alzheimer's disease

Alzheimer’s disease (AD) involves the neurotoxic self-assembly of a 40 and 42 residue peptide, Amyloid-β (Aβ). Inherited early-onset AD can be caused by single point mutations within the Aβ sequence, including Arctic (E22G) and Italian (E22K) familial mutants. These mutations are heterozygous, resulting in an equal proportion of the WT and mutant Aβ isoform expression. It is therefore important to understand how these mixtures of Aβ isoforms interact with each other and influence the kinetics and morphology of their assembly into oligomers and fibrils. Using small amounts of nucleating fibril seeds, here, we systematically monitored the kinetics of fibril formation, comparing self-seeding with cross-seeding behavior of a range of isoform mixtures of Aβ42 and Aβ40. We confirm that Aβ40(WT) does not readily cross-seed Aβ42(WT) fibril formation. In contrast, fibril formation of Aβ40(Arctic) is hugely accelerated by Aβ42(WT) fibrils, causing an eight-fold reduction in the lag-time to fibrillization. We propose that cross-seeding between the more abundant Aβ40(Arctic) and Aβ42(WT) may be important for driving early-onset AD and will propagate fibril morphology as indicated by fibril twist periodicity. This kinetic behavior is not emulated by the Italian mutant, where minimal cross-seeding is observed. In addition, we studied the cross-seeding behavior of a C-terminal-amidated Aβ42 analog to probe the coulombic charge interplay between Glu22/Asp23/Lys28 and the C-terminal carboxylate. Overall, these studies highlight the role of cross-seeding between WT and mutant Aβ40/42 isoforms, which can impact the rate and structure of fibril assembly.

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic β-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1–4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

Abstract PD8-07: A pro-metastatic alternative splicing program that drives breast cancer progression

Abstract Background: Breast cancer progression accompanies broad reprogramming of the alternative splicing landscape with functional implications for tumor growth and metastasis. However, the underlying regulatory pathways that are hijacked by cancer cells to drive pathological splicing programs remain largely unknown. Here, we have described a novel regulatory pathway, mediated by the splicing factor SNRPA1, that promotes breast cancer progression. Results: We used high-coverage RNA sequencing to compare alternative splicing patterns in highly metastatic breast cancer cells (MDA-LM2) to their less metastatic parental line (MDA-231). We identified a novel structural splicing enhancer (SSE) element that is enriched among the exons up-regulated in highly metastatic cells and showed that it is bound by the RNA-binding protein SNRPA1, which in turn acts as a splicing factor. We used loss-of-function and gain-of-function both in vitro and in vivo xenograft mouse models to establish SNRPA1 as a promoter of breast cancer cell invasion (~2-fold change in two independent cell lines, P=0.002) and metastasis (~5-10 fold change in lung colonization in two independent cell lines, P<0.01). We also identified specific splicing events downstream of SNRPA1 that are required for its pro-metastatic activity. Single and multivariate analyses of publicly available datasets established that the higher expression of SNRPA1 is significantly associated with metastatic progression (METABRIC: n=1456, P<1e-5; EXPO: n=243, P=0.002), as well as reduced metastasis-free (kmplot: n=1746, HR=1.4, P<1e-3) and disease-free survival (METABRIC: n=1979, HR=1.7, P<1e-6; multivariate: HR=1.3, P=0.01). Finally, we measured the expression of SNRPA1 and its target exon in 96 samples from normal tissue biopsy to stage IV metastatic disease and observed a concordant increase in the activity of this novel pro-metastatic splicing program (ANOVA: P<1e-10; U test stage IV to N: P<1e-3). Methods: We used a framework called ‘mixture of isoforms’ (MISO) to quantify changes in alternative splicing. We then applied iTEISER, a computational tool we have developed, to find cis-regulatory elements that drive alternative splicing patterns. We used bait RNA to co-precipitate and mass-spectrometry to identify the RNA-binding proteins that interact with SSEs. Lentiviral delivery of shRNAs (two independent hairpins) or transgenes to modulate SNRPA1 expression and RNA-seq was used to measure their regulatory consequences on splicing. For the resulting lines (both MDA-231 and HCC-1806 backgrounds), in vitro cancer cell invasion and in vivo lung colonization assays were also performed (n=5 mice in each arm). Log-rank test (univariate) and Cox Proportional Hazard Models (multivariate) were used to perform survival analyses in METABRIC and the kmplot aggregate dataset. Mann-Whitney or ANOVA was used to compare expression of SNRPA1 across samples stratified based on sub-type and tumor grade/stage in public datasets as well as our own measurements in clinical samples (n=96; 5 healthy, 23 stage I, 30 stage II, 29 stage III, and 9 stage IV). Conclusion: In sum, we have discovered a previously unknown regulatory pathway of RNA splicing that acts as a promoter of metastasis in breast, which may inform new therapeutic targets among the SNRPA1 regulon for adjuvant therapy. Citation Format: Hani Goodarzi, Lisa Fish, Bruce Culbertson. A pro-metastatic alternative splicing program that drives breast cancer progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-07.

Isolation, identification, and stability of Ficin 1c isoform from fig latex

Purified alkaline ficin isoform, identified as Ficin 1c regarding fig transcriptome, shows decreased stability compared to the ficin isoform mixture.

Production of Non-Toxic Biosurfactant – Surfactin – Through Microbial Fermentation of Biomass Hydrolysates for Industrial and Environmental Applications

Abstract The economically viable large-scale production of the pure isoforms of the surfactin biosurfactants, involving bacterial – Bacillus subtilis – fermentation of biomass hydrolysate feedstock, relies on the types of bacterial strains, optimization of the fermentation processing parameters, differences in the composition of the carbon and nitrogen in the bacterial media, and the chromatography techniques used for isolation of the isoforms. Here, we biosynthesized the surfactin isoforms in their mixture forms through fermentation of biomass hydrolysates at 2 wt.% carbohydrate content. The surfactin isoforms were assessed for their surface-active properties and toxicity. The enzyme hydrolysates considered were from switchgrass, soyhull (fiber), alfalfa, and bagasse. The isoform mixtures obtained after fermentation of the hydrolysates and, glucose as a control, were concentrated using chromatography columns, and characterized for molecular weights (MWs) and relative distribution using LCMS. The isoform mixtures, obtained in different fermenters (5- and 15-L) and, for different hydrolysates, invariably constituted 5 isoforms with MWs as 992.6, 1006.6, 1020.6, 1034.6, 1048.6, 1062.6 m/z amu, with their relative proportions as 6, 24, 35, 24, and 10 weight % respectively. The surface tension values of all these isoforms, in the absence of electrolytes and at 12 ppt salinity, were similar: 37 (pH 6.5) and 31 (pH 9.5) mN/m. Furthermore, the emulsification index values for the isoforms were also similar: Dispersant-to-Oil ratio as 1:20. The LC50 for Gulf killifish, Fundulus grandis for these surfactin isoforms ranged between 10 and 20 mg/L; a microbially-produced surfactin variant FA-Glu (Fatty acid Glutamate) was least toxic with LC50 at ∼100 mg/L. Thus, the surfactin synthesis approach adopted here suggested that pure (>95 wt.%) non-toxic isoforms of surfactin biosurfactants can be produced in the forms of their mixtures with surface-active properties similar to those of the pure forms of the surfactin isoforms.

Gene Expression and Missplicing in the Corneal Endothelium of Patients With a TCF4 Trinucleotide Repeat Expansion Without Fuchs' Endothelial Corneal Dystrophy.

PurposeCTG trinucleotide repeat (TNR) expansion in an intron of the TCF4 gene is the most common genetic variant associated with Fuchs' endothelial corneal dystrophy (FECD). Although several mechanisms have been implicated in the disease process, their exact pathophysiologic importance is unclear. To understand events leading from TCF4 TNR expansion to disease phenotype, we characterized splicing, gene expression, and exon sequence changes in a rare cohort of patients with TNR expansions but no phenotypic FECD (RE+/FECD−).MethodsCorneal endothelium and blood were collected from patients undergoing endothelial keratoplasty for non-FECD corneal edema. Total RNA was isolated from corneal endothelial tissue (n = 3) and used for RNASeq. Gene splicing and expression was assessed by Mixture of Isoforms (MISO) and MAP-RSeq software. Genomic DNA was isolated from blood mononuclear cells and used for whole genome exome sequencing. Base calling was performed using Illumina's Real-Time Analysis.ResultsThree genes (MBNL1, KIF13A, AKAP13) that were previously identified as misspliced in patients with a CTG TNR expansion and FECD disease (RE+/FECD+) were found normally spliced in RE+/FECD− samples. Gene expression differences in pathways associated with the innate immune response, cell signaling (e.g., TGFβ, WNT), and cell senescence markers were also identified between RE+/FECD− and RE+/FECD+ groups. No consistent genetic variants were identified in RE+/FECD− patient exomes.ConclusionsIdentification of novel splicing patterns and differential gene expression in RE+/FECD− samples provides new insights and more relevant gene targets that may be protective against FECD disease in vulnerable patients with TCF4 CTG TNR expansions.

Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX.

Leptospira interrogans genome is predicted to encode multiple isoforms of caseinolytic proteases (ClpP1 and ClpP2). The ClpP proteins with the aid of its ATPase chaperone are known to be involved in establishing cellular proteostasis and have emerged as a target for developing new antibiotics. We report the molecular characterization of recombinant ClpP1 (rClpP1) and rClpP2 of Leptospira along with its ATPase chaperone rClpX. The two isoforms of rClpPs when coupled together in an equivalent concentration exhibit optimum activity on small fluorogenic peptide substrates, whereas the pure rClpP isoforms are enzymatically inactive. Isothermal titration calorimetry analysis suggests that the two rClpP isoforms bind each other moderately in a 1:1 stoichiometry with a dissociation constant of 2.02 ± 0.1 μM at 37 °C and is thermodynamically favored. Size exclusion chromatography fractionates the majority of pure rClpP1 at ≥308 kDa (14–21-mer) and the pure rClpP2 at 308 kDa (tetradecamer), whereas the functionally active rClpP isoform mixture fractionates as a tetradecamer. The distinct and unprecedented oligomeric form of rClpP1 was also evident through native-gel and dynamic light scattering. Moreover, the rClpP isoform mixture formed after the site-directed mutation of either or both the isoforms at one of the catalytic triad residues (Ser 98/97 to Ala 98/97) resulted in the complete loss of protease activity. The rClpP isoform mixture gets stimulated to degrade the casein substrate in the presence of rClpX and in an energy-dependent manner. On the contrary, pure rClpP1 or the rClpP2 isoform in association with rClpX are incapable of forming operative protease. The reported finding suggests that in Leptospira, the enzymatic activity of the rClpP protease complex in the presence or absence of cochaperone is performed solely by the tetradecamer structure which is hypothesized to be composed of 2-stacked ClpP heptameric rings, wherein each ring is a homo-oligomer of ClpP1 and ClpP2 subunits. Understanding the activities and regulation principle of multi-isoforms of ClpP in pathogenic bacteria may aid in intervening disease outcomes particularly to the co-evolving antibiotic resistance strains.

Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology.

Micromolar concentrations of hyperactive antifreeze proteins (AFPs) from insects can prevent aqueous solutions from freezing down to at least −6 °C. To explore cryopreservation of cells, tissues and organs at these temperatures without ice formation, we have developed a protocol to reliably produce ultrapure Tenebrio molitor AFP from cold-acclimated beetle larvae reared in the laboratory. The AFP was prepared from crude larval homogenates through five cycles of rotary ice-affinity purification, which can be completed in one day. Recovery of the AFP at each step was >90% and no impurities were detected in the final product. The AFP is a mixture of isoforms that are more active in combination than any one single component. Toxicity testing of the purified AFP in cell culture showed no inhibition of cell growth. The production process can easily be scaled up to industrial levels, and the AFP used in cryobiology applications was recovered for reuse in good yield and with full activity.

Mo-CBP3, a 2S albumin from Moringa oleifera, is a complex mixture of isoforms that arise from different post-translational modifications

Mo-CBP3 is a chitin-binding 2S albumin from Moringa oleifera. This seed storage protein is resistant to thermal denaturation and shows biological activities that might be of practical use, such as antifungal properties against Candida sp., a pathogen that causes candidiasis, and against Fusarium solani, a soil fungus that can cause diseases in plants and humans. Previous work has demonstrated that Mo-CBP3 is a mixture of isoforms encoded by members of a small multigene family. Mature Mo-CBP3 is a small protein (∼14 kDa), constituted by a small chain of approximately 4 kDa and a large chain of 8 kDa, which are held together by disulfide bridges. However, a more comprehensive picture on the spectrum of Mo-CBP3 isoforms which are found in mature seeds, is still lacking. In this work, genomic DNA fragments were obtained from M. oleifera leaves, cloned and completely sequenced, thus revealing new genes encoding Mo-CBP3. Moreover, mass spectrometry analysis showed that the mature protein is a complex mixture of isoforms with a remarkable number of molecular mass variants. Using computational predictions and calculations, most (∼86%) of the experimentally determined masses were assigned to amino acid sequences deduced from DNA fragments. The results suggested that the complex mixture of Mo-CBP3 isoforms originates from proteins encoded by closely related genes, whose products undergo different combinations of distinct post-translational modifications, including cleavage at the N- and C-terminal ends of both subunits, cyclization of N-terminal Gln, as well as Pro hydroxylation, Ser/Thr phosphorylation, and Met oxidation.

Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa.

Odorant-binding proteins (OBP) are secreted in the nasal mucus at the vicinity of olfactory receptors (ORs). They act, at least, as an interface between hydrophobic and volatile odorant molecules and the hydrophilic medium bathing the ORs. They have also been hypothesized to be part of the molecular coding of odors and pheromones, by forming specific complexes with odorant molecules that could ultimately stimulate ORs to trigger the olfactory transduction cascade. In a previous study, we have evidenced that pig olfactory secretome was composed of numerous olfactory binding protein isoforms, generated by O-GlcNAcylation and phosphorylation. In addition, we have shown that recombinant OBP (stricto sensu) produced in yeast is made up of a mixture of isoforms that differ in their phosphorylation pattern, which in turn determines binding specificity. Taking advantage of the high amount of OBP secreted by a single animal, we performed a similar study, under exactly the same experimental conditions, on native isoforms isolated from pig, Sus scrofa, nasal tissue. Four fractions were obtained by using strong anion exchange HPLC. Mapping of phosphorylation and O-GlcNAcylation sites by CID-nanoLC-MS/MS allowed unambiguous localization of phosphosites at S13 and T122 and HexNAc sites at S13 and S19. T112 or T115 could also be phosphorylated. BEMAD analysis suggested extra phosphosites located at S23, S24, S41, S49, S57, S67, and T71. Due to the very low stoichiometry of GlcNAc-peptides and phosphopeptides, these sites were identified on total mixture of OBP isoforms instead of HPLC-purified OBP isoforms. Nevertheless, binding properties of native OBP isoforms to specific ligands in S. scrofa were monitored by fluorescence spectroscopy. Recombinant phosphorylated OBP-Pichia isoforms bind steroids and fatty acids with slight differences. Native isoforms, that are phosphorylated but also O-GlcNAcylated show radically different binding affinities for the same compounds, which strongly suggests that O-GlcNAcylation increases the binding specificity of OBP isoforms. These findings extend the role of O-GlcNAc in regulating the function of proteins involved in many mechanisms of metabolic homeostasis, including extracellular signaling in olfaction. Data is available via ProteomeXChange with identifier PXD011371.

β-Lactoglobulin as a potential carrier for bioactive molecules

In this study, the interaction and binding behavior of anesthetic tetracaine (TET) with bovine β-lactoglobulin (LGB) isoform A and a mixture of isoforms A and B were investigated under varying environmental conditions (pH, ionic strength, concentration, LGB-TET complex molar ratio). A wide range of analytical techniques (dynamic light scattering (DLS), electrophoretic mobility, UV–Vis spectroscopy, circular dichroism (CD), quartz crystal microbalance (QCM-D) were used to analyze the physicochemical properties of the complexes in bulk solution and on the surface of gold. The experiments revealed that TET, which is amphiphilic, could bind with LGB not only in the β-barrel but also onto the surface. The zeta potential of the LGB becomes more positively charged upon interaction with TET due to electrostatic interaction of the amino group present in the TET structure. Changes in the zeta potential values are mostly visible above pH 6 for all tested systems. CD spectra show that interaction with the ligand does not change the secondary structure of the protein. The physicochemical properties of LGB-TET complex were examined in an adsorbed state on a gold surface using the QCM-D method. Additionally, molecular docking was used to evaluate the most likely binding site for TET with LGB.

Enantioselectivity of 2,2',3,5',6-Pentachlorobiphenyl (PCB 95) Atropisomers toward Ryanodine Receptors (RyRs) and Their Influences on Hippocampal Neuronal Networks.

Nineteen ortho-substituted PCBs are chiral and found enantioselectively enriched in ecosystems. Their differential actions on biological targets are not understood. PCB 95 (2,2',3,5',6-pentachlorobiphenyl), a chiral PCB of current environmental relevance, is among the most potent toward modifying ryanodine receptors (RyR) function and Ca2+ signaling. PCB 95 enantiomers are separated and assigned aR- and aS-PCB 95 using three chiral-column HPLC and circular dichroism spectroscopy. Studies of RyR1-enriched microsomes show aR-PCB 95 with >4× greater potency (EC50 = 0.20 ± 0.05 μM), ∼ 1.3× higher efficacy (Bmax = 3.74 ± 0.07 μM) in [3H]Ryanodine-binding and >3× greater rates (R = 7.72 ± 0.31 nmol/sec/mg) of Ca2+ efflux compared with aS-PCB 95, whereas racemate has intermediate activity. aR-PCB 95 has modest selectivity for RyR2, and lower potency than racemate toward the RyR isoform mixture in brain membranes. Chronic exposure of hippocampal neuronal networks to nanomolar PCB 95 during a critical developmental period shows divergent influences on synchronous Ca2+ oscillation (SCO): rac-PCB 95 increasing and aR-PCB 95 decreasing SCO frequency at 50 nM, although the latter's effects are nonmonotonic at higher concentration. aS-PCB95 shows the greatest influence on inhibiting responses to 20 Hz electrical pulse trains. Considering persistence of PCB 95 in the environment, stereoselectivity toward RyRs and developing neuronal networks may clarify health risks associated with enantioisomeric enrichment of PCBs.

Quantification of Total Vitamin-D-Binding Protein and the Glycosylated Isoforms by Liquid Chromatography-Isotope Dilution Mass Spectrometry.

Vitamin-D-binding protein (VDBP), a transporter of 25-hydroxyvitamin D metabolites, has three common isoforms. The relationship of the isoforms and their glycosylation state with various diseases has been under recent examination. In this work, liquid chromatography coupled to isotope dilution mass spectrometry was evaluated for quantification of VDBP, the three common isoforms, and total glycosylation. Protocols using guanidine, urea, RapiGest, trifluoroethanol, or tris buffer were also evaluated for optimal tryptic digestion. Differences in peptide release were detected between purified and plasma VDBP; however, for both protein sources, ELPEHTVK, TSALSAK, and VLEPTLK concentrations were reproducible between most protocols tested. The isoform-specific peptides, LPDATPK, LPDATPTELAK, and LPEATPTELAK, were optimally released when TFE was added to plasma. The total VDBP concentration calculated from the three shared peptides resulted in 97.6% accuracy compared with the concentration from amino acid analysis. Glycosylation of VDBP was also calculated for purified protein and donor samples using the ratio of the isoform-specific peptide(s) to the total protein concentration. Glycosylation of purified VDBP was found to be 99.5-111.1% the value determined by semiquantitative analysis of the intact protein by LC-MS. This approach may be used to quantify other samples containing a mixture of isoforms and post-translational modifications.

Structural characterization of two isolectins from the marine red alga Solieria filiformis (Kützing) P.W. Gabrielson and their anticancer effect on MCF-7 breast cancer cells

As described in the literature, Solieria filiformis lectin (SfL) from the marine red alga S. filiformis was found to have antinociceptive and anti-inflammatory effects. In this study, we characterized two SfL variants, SfL-1 and SfL-2, with molecular mass of 27,552Da and 27,985Da, respectively. The primary structures of SfL-1 and SfL-2 consist of four tandem-repeat protein domains with 67 amino acids each. SfL-1 and -2 showed high similarity to OAAH-family lectins. 3D structure prediction revealed that SfL-1 and -2 are composed of two β-barrel-like domains formed by five antiparallel β-strands, which are connected by a short peptide linker. Furthermore, the mixture of isoforms (SfLs) showed anticancer effect against MCF-7 cells. Specifically, SfLs inhibited 50% of viability in MCF-7 cells after treatment at 125μg.mL−1, while the inhibition of Human Dermal Fibroblasts (HDF) was 34% with the same treatment. Finally, 24h after treatment, 25% of MCF-7 cells were in early apoptosis and 35% in late apoptosis. Evaluation of pro- and anti-apoptotic gene expression of MCF-7 cells revealed that SfLs induced caspase-dependent apoptosis within 24h.

Antimicrobial activity of lysozyme isoforms: Key molecular features.

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

Olfactory effects of a hypervariable multicomponent pheromone in the red-legged salamander, Plethodon shermani.

Chemical communication via chemosensory signaling is an essential process for promoting and modifying reproductive behavior in many species. During courtship in plethodontid salamanders, males deliver a mixture of non-volatile proteinaceous pheromones that activate chemosensory neurons in the vomeronasal epithelium (VNE) and increase female receptivity. One component of this mixture, Plethodontid Modulating Factor (PMF), is a hypervariable pheromone expressed as more than 30 unique isoforms that differ between individual males—likely driven by co-evolution with female receptors to promote gene duplication and positive selection of the PMF gene complex. Courtship trials with females receiving different PMF isoform mixtures had variable effects on female mating receptivity, with only the most complex mixtures increasing receptivity, such that we believe that sufficient isoform diversity allows males to improve their reproductive success with any female in the mating population. The aim of this study was to test the effects of isoform variability on VNE neuron activation using the agmatine uptake assay. All isoform mixtures activated a similar number of neurons (>200% over background) except for a single purified PMF isoform (+17%). These data further support the hypothesis that PMF isoforms act synergistically in order to regulate female receptivity, and different putative mechanisms are discussed.

Insights from native mass spectrometry and ion mobility-mass spectrometry for antibody and antibody-based product characterization.

Over the past 15 years, monoclonal antibodies (mAbs) have emerged as the most successful class of therapeutics. Their specific structural and functional properties make them highly effective treatments for various diseases. Most therapeutic mAbs are based on chimeric, humanized or human G immunoglobulins (IgGs) selected from three isotypes (1, 2 and 4). IgGs are large and highly complex multimeric glycoproteins. They are constituted of a mixture of isoforms including macro and micro-variants that must be extensively characterized prior to their investigation as a drug candidate in clinical trials. The IgG backbone is also used to design more potent but also more complex biopharmaceuticals such as antibody-drug conjugates, bispecific antibodies, Fc-fusion proteins, and antibody mixtures to name a few. Mass spectrometric approaches in combination with electrophoretic and chromatographic separation methods play a central role in the analytical and structural multi-level (top, middle and bottom) characterization of these compounds. Importantly, techniques allowing the characterization of intact mAbs and related products under non-denaturing conditions are attracting increasing interest. Here, we review the current state of the art in native mass spectrometry and ion mobility methods for the characterization of mAbs and mAb-based products.