Structural basis for the inhibition of ßFXIIa by garadacimab.

Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (ßFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to ßFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-ßFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of ßFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-ßFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.

HDX-MS study on garadacimab binding to activated FXII reveals potential binding interfaces through differential solvent exposure.

Hageman factor (FXII) is an essential component in the intrinsic coagulation cascade and a therapeutic target for the prophylactic treatment of hereditary angioedema (HAE). CSL312 (garadacimab) is a novel high-affinity human antibody capable of blocking activated FXII activity that is currently undergoing Phase 3 clinical trials in HAE. Structural studies using hydrogen/deuterium exchange coupled to mass spectrometry revealed evidence of interaction between the antibody and regions surrounding the S1 specificity pocket of FXII, including the 99-loop, 140-loop, 180-loop, and neighboring regions. We propose complementarity-determining regions (CDRs) in heavy-chain CDR2 and CDR3 as potential paratopes on garadacimab, and the 99-loop, 140-loop, 180-loop, and 220-loop as binding sites on the beta chain of activated FXII (ß-FXIIa).

Sialylation-dependent pharmacokinetics and differential complement pathway inhibition are hallmarks of CR1 activity in vivo.

Human Complement Receptor 1 (HuCR1) is a potent membrane-bound regulator of complement both in vitro and in vivo, acting via interaction with its ligands C3b and C4b. Soluble versions of HuCR1 have been described such as TP10, the recombinant full-length extracellular domain, and more recently CSL040, a truncated version lacking the C-terminal long homologous repeat domain D (LHR-D). However, the role of N-linked glycosylation in determining its pharmacokinetic (PK) and pharmacodynamic (PD) properties is only partly understood. We demonstrated a relationship between the asialo-N-glycan levels of CSL040 and its PK/PD properties in rats and non-human primates (NHPs), using recombinant CSL040 preparations with varying asialo-N-glycan levels. The clearance mechanism likely involves the asialoglycoprotein receptor (ASGR), as clearance of CSL040 with a high proportion of asialo-N-glycans was attenuated in vivo by co-administration of rats with asialofetuin, which saturates the ASGR. Biodistribution studies also showed CSL040 localization to the liver following systemic administration. Our studies uncovered differential PD effects by CSL040 on complement pathways, with extended inhibition in both rats and NHPs of the alternative pathway compared with the classical and lectin pathways that were not correlated with its PK profile. Further studies showed that this effect was dose dependent and observed with both CSL040 and the full-length extracellular domain of HuCR1. Taken together, our data suggests that sialylation optimization is an important consideration for developing HuCR1-based therapeutic candidates such as CSL040 with improved PK properties and shows that CSL040 has superior PK/PD responses compared with full-length soluble HuCR1.

A novel soluble complement receptor 1 fragment with enhanced therapeutic potential.

Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders.

Natural Mutagenesis-Enabled Global Proteomic Study of Metabolic and Carbon Source Implications in Mutant Thermoacidophillic Archaeon Sulfolobus solfataricus PBL2025.

The thermoacidophilic crenarchaeon Sulfolobus solfataricus has been widely used as a model organism for archaeal systems biology research. Investigation using its spontaneous mutant PBL2025 provides an effective metabolic baseline to study subsequent mutagenesis-induced functional process shifts as well as changes in feedback inhibitions. Here, an untargeted metabolic investigation using quantitative proteomics and metabolomics was performed to correlate changes in S. solfataricus strains P2 against PBL2025 and under both glucose and tryptone. The study is combined with pathway enrichment analysis to identify prominent proteins with differential stoichiometry. Proteome level quantification reveals that over 20% of the observed overlapping proteome is differentially expressed under these conditions. Metabolic-induced differential expressions are observed along the central carbon metabolism, along with 12 other significantly regulated pathways. Current findings suggest that PBL2025 is able to compensate through the induction of carbon metabolism, as well as other anabolic pathways such as Val, Leu and iso-Leu biosynthesis. Studying protein abundance changes after changes in carbon sources also reveals distinct differences in metabolic strategies employed by both strains, whereby a clear down-regulation of carbohydrate and nucleotide metabolism is observed for P2, while a mixed response through down-regulation of energy formation and up-regulation of glycolysis is observed for PBL2025. This study contributes, to date, the most comprehensive network of changes in carbohydrate and amino acid pathways using the complementary systems biology observations at the protein and metabolite levels. Current findings provide a unique insight into molecular processing changes through natural (spontaneous) metabolic rewiring, as well as a systems biology understanding of the metabolic elasticity of thermoacidophiles to environmental carbon source change, potentially guiding more efficient directed mutagenesis in archaea.

Quantitative analysis of UV-A shock and short term stress using iTRAQ, pseudo selective reaction monitoring (pSRM) and GC-MS based metabolite analysis of the cyanobacterium Nostoc punctiforme ATCC 29133.

A quantitative proteomics and metabolomics analysis was performed using iTRAQ, HPLC and GC-MS in the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 to understand the effect of short and long term UV-A exposure. Changes in the proteome were measured for short-term stress (4-24h) using iTRAQ. Changes in the photosynthetic pigments and intracellular metabolites were observed at exposures of up to 7days (pigments) and up to 11days (intracellular metabolites). To assess iTRAQ measurement quality, pseudo selected reaction monitoring (pSRM) was used, with this confirming underestimation of protein abundance levels by iTRAQ. Our results suggest that short term UV-A radiation lowers the abundance of PS-I and PS-II proteins. We also observed an increase in abundance of intracellular redox homeostasis proteins and plastocyanin. Additionally, we observed statistically significant changes in scytonemin, Chlorophyll A, astaxanthin, zeaxanthin, and ß-carotene. Assessment of intracellular metabolites showed significant changes in several, suggesting their potential role in the Nostoc's stress mitigation strategy. Cyanobacteria under UV-A radiation have reduced growth due to intensive damage to essential functions, but the organism shows a defense response by remodeling bioenergetics pathway, induction of the UV protection compound scytonemin and increased levels of proline and tyrosine as a mitigation response. BIOLOGICAL SIGNIFICANCE: The effect of UV-A radiation on the proteome and intracellular metabolites of N. punctiforme ATCC 29133 including photosynthetic pigments has been described. We also verify the expression of 13 iTRAQ quantified protein using LC-pSRM. Overall we observed that UV-A radiation has a drastic effect on the photosynthetic machinery, photosynthetic pigments and intracellular amino acids. As a mitigation strategy against UV-A radiation, proline, glycine, and tyrosine were accumulated.

Application of the CIRAD mass spectrometry approach for lysine acetylation site discovery.

Mass spectrometry (MS)-based methods typically assess acetylation by detection of a diagnostic ion at 126.1 m/z, corresponding to the immonium ion of acetyl-lysine -NH(3), which is generated by collisionally induced dissociation. A novel implementation of this approach, based on the accurate mass and retention time technique, couples high mass resolution measurement with rapid cycling between low and elevated collision energies to generate intact and fragment high-resolution mass spectra. This allows acetyl lysine diagnostic ions at 126.1 m/z to be monitored and aligned to the precursor m/z based on retention time profile. The technique is termed Collisionally Induced Release of Acetyl Diagnostic. Sequence information is also obtained for acetylation site assignment. This technique to identify acetylation species is information independent as it does not require the sequence of the protein/peptides to identify acetylation, and thus complementary to data-dependent methods. It is suitable for analysis of acetylated peptides, or proteins enriched by immunoprecipitation with acetyl lysine-specific antibodies.

The quantitative proteomic response of Synechocystis sp. PCC6803 to phosphate acclimation.

BACKGROUND: Inorganic phosphate (Pi) is a critical nutrient for all life and is periodically limiting in marine and freshwater provinces, yet little is understood how organisms acclimate to fluctuations in Pi within their environment. To investigate whole cell adaptation, we grew Synechocystis sp. PCC6803, a model freshwater cyanobacterium, in 3%, and 0.3% inorganic phosphate (Pi) media. The cells were allowed to acclimate over 60 days, and cells were harvested for quantitative high throughput mass spectrometry-based proteomics using the iTRAQ™ labelling technology. RESULTS: In total, 120 proteins were identified, and 52 proteins were considered differentially abundant compared to the control. Alkaline phosphatase (APase) activities correlated significantly (p < 0.05) with observed relative PhoA abundances. PstS1 and PstS2 were both observed, yet PstS1 was not differentially more abundant than the control. Phycobilisome protein abundances appeared to be coordinated, and are significantly less abundant in 0.3% Pi than 3% Pi cultures. Also, the central metabolic cell function appears to have shifted towards the production of (NADPH) reducing energy and nucleotide sugars. CONCLUSIONS: This acclimation response bears strong similarity to the previously reported response to nitrogen deprivation within Synechocystis sp. PCC 6803. However, it also demonstrates some characteristics of desiccation stress, such as the regulation of fatty acids and increased abundance of rehydrin in the 3% Pi culture.

Quantitative proteomic analysis of the exoelectrogenic bacterium Arcobacter butzleri ED-1 reveals increased abundance of a flagellin protein under anaerobic growth on an insoluble electrode.

Exoelectrogens have the ability to generate electricity in mediator-less microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigate the anode-specific responses of Arcobacter butzleri ED-1, the first identified exoelectrogenic Epsilonproteobacterium. iTRAQ and 2D-LC MS/MS driven proteomics were used to compare protein abundances in A. butzleri ED-1 when generating an electronegative potential (-225 mV) in an anaerobic half-cell - either growing as an electrogenic biofilm or suspended in the liquid medium - versus a microaerobic culture. This is the first quantitative proteomic study concentrating on growth of an exoelectrogen during current generation. From 720 proteins identified and quantified (soluble and insoluble sub-proteomes), statistical analysis reveals 75 differentially-expressed proteins. This dataset was enriched in proteins regulating energy and intermediary metabolism, electron and protein transport. Flagellin up-regulation was concomitant with electron transport in the anodic cells, while decreased abundance of a methyl-accepting chemotaxis protein suggested that flagella were involved in communication with the anode surface and electrogenesis, rather than motility. Two novel cytochromes potentially related to electron transport were up-regulated in anaerobic cultures. We demonstrate that employing an insoluble extracellular electron acceptor for anaerobic growth regulates multiple proteins involved in cell surface properties, electron transport and the methylcitrate cycle.

An insight into iTRAQ: where do we stand now?

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.

iTRAQ-based quantitative proteomic analysis of Gloeothece sp. PCC 6909: Comparison with its sheathless mutant and adaptations to nitrate deficiency and sulfur limitation.

Gloeothece sp. PCC 6909 is a unicellular N(2)-fixing cyanobacterium with a well defined and highly developed sheath surrounding its cells. A sheathless mutant of this strain was previously obtained by chemical mutagenesis and, although lacking the sheath, it releases large amounts of polysaccharides into the culture medium. To provide a global understanding on the metabolic differences between the two phenotypes, the proteomes of the wild type and mutant were analyzed using a cross-species proteomics approach coupled with iTRAQ isobaric tagging technology, since their genome sequences are not yet available. Effects arising from the presence/absence of nitrate and sulfur are presented as two metabolically directed follow-up iTRAQ studies. These nutrients are believed to play a major role in Gloeothece's metabolism, including the production of extracellular polymeric substances - EPS. 454, 124, and 53 proteins were identified and reliably quantified using homology anchoring approaches for iTRAQ previously described. The results obtained strongly suggest that the chemical mutagenesis affected the regulation of a number of key cellular processes, as revealed by the significant fold changes observed for proteins covering a large spectrum of functional groups. Moreover, they provide new insights on the adaptations of Gloeothece cells to nitrate-deficiency and sulfur-limitation.

Minimising iTRAQ ratio compression through understanding LC-MS elution dependence and high-resolution HILIC fractionation.

Application of iTRAQ-based workflows for protein profiling has become widespread. Concomitantly, the idiosyncratic limitations of iTRAQ, such as its tendency to underestimate quantifications, have been studied and recognised. This report shows that the influence of ratio compression and limiting transmission in iTRAQ MS/MS in high-complexity mixtures (iTRAQ-labelled lysates) can be partly alleviated using high-resolution sample fractionation. Here, we also investigate in greater detail the dependency of iTRAQ quantification on the dynamics of online chromatography in low-complexity mixtures (iTRAQ-labelled standards). These findings will allow more efficient strategies to be designed for iTRAQ proteomics, alleviating iTRAQ underestimation and thus facilitating the detection of subtle abundance changes.

Improving N-glycosylation efficiency in Escherichia coli using shotgun proteomics, metabolic network analysis, and selective reaction monitoring.

Recently, the prospect of using Escherichia coli as a host for human glycoprotein production has increased due to detailed characterization of the prokaryotic N-glycosylation process and the ability to transfer the system into this bacterium. Although functionality of the native Campylobacter jejuni N-glycosylation system in E. coli has been demonstrated, the efficiency of the process using the well-characterized C. jejuni glycoprotein AcrA, was found to be low at 13.4±0.9% of total extracted protein. A combined approach using isobaric labeling of peptides and probability-based network analysis of metabolic changes was applied to forward engineer E. coli to improve glycosylation efficiency of AcrA. Enhancing flux through the glyoxylate cycle was identified as a potential metabolic manipulation to improve modification efficiency and was achieved by increasing the expression of isocitrate lyase. While the overall recombinant protein titre did not change significantly, the amount of glycosylated protein increased by approximately 300%.

Metabolic adaptations in a H2 producing heterocyst-forming cyanobacterium: potentials and implications for biological engineering.

Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the ability to fix atmospheric nitrogen and photoproduce hydrogen through the enzyme nitrogenase. The H(2) produced is reoxidized by an uptake hydrogenase. Inactivation of the uptake hydrogenase in N. punctiforme leads to increased H(2) release but unchanged rates of N(2) fixation, indicating redirected metabolism. System-wide understanding of the mechanisms of this metabolic redirection was obtained using complementary quantitative proteomic approaches, at both the filament and the heterocyst level. Of the total 1070 identified and quantified proteins, 239 were differentially expressed in the uptake hydrogenase mutant (NHM5) as compared to wild type. Our results indicate that the inactivation of uptake hydrogenase in N. punctiforme changes the overall metabolic equilibrium, affecting both oxygen reduction mechanisms in heterocysts as well as processes providing reducing equivalents for metabolic functions such as N(2) fixation. We identify specific metabolic processes used by NHM5 to maintain a high rate of N(2) fixation, and thereby potential targets for further improvement of nitrogenase based H(2) photogeneration. These targets include, but are not limited to, components of the oxygen scavenging capacity and cell envelope of heterocysts and proteins directly or indirectly involved in reduced carbon transport from vegetative cells to heterocysts.

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle.

Nuclear proteome dynamics in differentiating embryonic carcinoma (NTERA-2) cells.

The use of stem cells for generating cell types suitable for therapy is dependent on understanding the mechanisms, and identifying biomarkers, that control cell fate into different lineages. In this study, we aimed to characterize the nuclear protein dynamics of NTERA-2 cells undergoing retinoic acid-induced differentiation. We focused specifically on the first six days of differentiation, to provide insight into the earliest differentiation events, and employed techniques to specifically monitor the nuclear proteome. Well-characterized gene expression markers were used to precisely stage cell differentiation across the experimental time course. A combination of the novel iTRAQ and ExacTag labeling technologies, together with LC-ESI tandem mass spectrometry, were then used to accurately measure nuclear protein expression changes occurring within these differentiation-staged cells. We report proteins that showed significantly altered expression over the first 6 days of differentiation. Extensive bioinformatic analysis was undertaken, resulting in the construction of a novel interactome network, which revealed the temporal dynamics of the nuclear protein network in the context of neuronal differentiation.

Balancing robust quantification and identification for iTRAQ: application of UHR-ToF MS.

iTRAQ reagents allow the simultaneous multiplex identification and quantification of a large number of proteins. Success depends on effective peptide fragmentation in order to generate both peptide sequence ions (higher mass region, 150-2200 m/z) and reporter ions (low mass region, 113-121 m/z) for protein identification and relative quantification, respectively. After collision-induced dissociation, the key requirements to achieve a good balance between the high and low m/z ions are effective ion transmission and detection across the MS/MS mass range, since the ion transmission of the higher m/z range competes with that of the low m/z range. This study describes an analytical strategy for the implementation of iTRAQ on maXis UHR-Qq-ToF instruments, and discusses the impact of adjusting the MS/MS ion transmission parameters on the quality of the overall data sets. A technical discussion highlights a number of maXis-specific parameters, their impact of quantification and identification, and their cross-interactions.

Systems biology meets synthetic biology: a case study of the metabolic effects of synthetic rewiring.

We present a systems biology approach to study the global metabolic effects of the insertion of synthetic circuits in a cellular chassis. Our approach combines high-throughput proteomics with the MMG probabilistic tool, which integrates the data with the metabolic circuit's topology. We present a theoretical analysis of the foundations of our approach, as well as experimental results on a mutant strain of Escherichia coli where a light-receptor circuit was inserted and coupled with lactose metabolism. Our results show that the systems approach manages to extract meaningful information from the proteomic data that cannot be recovered by naive thresholding of the data. This tool can be used to characterise the relationship between new circuits and chassis in synthetic biology applications.

iTRAQ underestimation in simple and complex mixtures: "the good, the bad and the ugly".

The increasing popularity of iTRAQ for quantitative proteomics applications makes it necessary to evaluate its relevance, accuracy, and precision for biological interpretation. Here, we have assessed (a) the accuracy and precision of iTRAQ quantification in a controlled experimental setup, using low- and high-complexity protein mixtures; and (b) the potential pitfalls that hamper the applicability and attainable dynamic range of iTRAQ: isotopic contamination, background interference, and signal-to-noise ratio. Our data suggest greater dynamic crosstalk between interfering factors affecting underestimations, and that these interferences were largely scenario-specific, dependent on sample complexity. The good is the potential for iTRAQ to provide accurate quantification spanning 2 orders of magnitude. This potential is however limited by two factors. (1) The bad: the existence of isotopic impurities that can be corrected for; provided accurate isotopic factors are at one's disposal. (2) The ugly: we demonstrate here the interference of mixed MS/MS contribution occurring during precursor selection, an issue that is currently very difficult to minimize. In light of our results, we propose a list of advice for iTRAQ data analysis that could routinely ameliorate quantitative interpretation of proteomic data sets.

Current trends in high throughput proteomics in cyanobacteria.

Advancements in genome sequencing and high throughput proteomics of cyanobacterial strains led to 13 published reports, from a small number of laboratories. These successful studies focused on Synechocystis, Nostoc and Anabaena strains, prochlorococcus, and halotolerant Euhalothece. The implications of emerging quantitative aspects developed and applied in these large-scale studies are assessed in the wake of advanced cyanobacterial research. Furthermore, contributions from traditional and early high throughput analysis of cyanobacterial proteomics are compared and summarised. Finally, opinions are provided to link both the trends and the future challenges. This review aims to push the synergy between proteomics and cyanobacterial research to improve both the technical and biological significance.