Biology subjects

Nyman, T. A.

Publications and source records attributed to Nyman, T. A..

14 recordsLinked to original sources

bioRxiv2026

Rapid plastid isolation reveals the chloroplast proteome and structures of the chlororibosome large subunit and RuBisCO in Marchantia polymorpha

Plastids house the biology of eukaryotic photosynthesis. While 1000s of plastid genomes have been sequenced, the availability of less than ten proteomes and only two species with full 70S plastid ribosomal structures limit our understanding of plant evolution. We optimized a protocol for the rapid isolation of Marchantia polymorpha plastids that provides a highly enriched and intact organelle fraction from gradient volumes as little as 2 mL. The approach was successfully applied to six other species. Focusing on M. polymorpha, we determined the proteome of the plastid fraction, identifying 1337 nuclear-encoded proteins with a high confidence, where 83% belong to orthologs shared with angiosperms. We further isolated protein complexes by RNA affinity purification using poly-lysine and provide the high-resolution structures of the 50S subunit of the chloroplast ribosome and RuBisCO using cryogenic EM and image reconstruction to 2.23 and 2.12 [A] resolution, respectively. For plastids, our data show that the genome reduction event experienced by the bryophyte common ancestor has had little impact on the organelles complexity and they underscore a high level of structural conservation of key components. Our data provide novel resources to explore the functional evolution of plastid proteomes and major macromolecular complexes of cyanobacterial origin.

plant biology↗

bioRxiv2026

Carbon source-dependent metabolic states govern redox homeostasis and cofactor biosynthesis in Propionibacterium freudenreichii

Microbial adaptation to fluctuating nutrient and oxygen conditions requires coordinated regulation of metabolic networks to maintain redox homeostasis within physicochemical and energetic constraints. While oxygen-dependent responses in Propionibacterium freudenreichii (PFR) have been characterized at the transcriptomic level, the role of carbon source in defining system-level metabolic states remains unclear. Here, we investigated carbon source-dependent metabolic reprogramming and cofactor biosynthesis in PFR strain DSM 20271 using label-free quantitative proteomics integrated with physiological and metabolite analyses. Distinct carbon sources defined discrete metabolic states shaped by redox balance and flux distribution. Lactate supported a comparatively balanced physiological state characterized by enhanced respiratory metabolism, amino acid biosynthesis, and riboflavin metabolism, enabling high specific vitamin B12 yields ([~]100 {micro}g g-1 wet biomass). In contrast, hexose metabolism (glucose and fructose) imposed a redox-constrained state marked by upregulation of transport systems, glycolysis, and the pentose phosphate pathway, resulting in increased biomass but reduced biosynthetic efficiency. A defining feature of the hexose-driven state was activation of aspartate metabolism. Proteomic and metabolite data, together with functional assays, support a model in which aspartate is converted to fumarate and subsequently reduced to succinate, providing an alternative electron sink that facilitates NADH reoxidation under redox-constrained conditions. Together, these findings establish that carbon source shapes physiological state through flux distribution, redox homeostasis, and resource allocation, with cofactor biosynthesis emerging as a system-level property rather than a simple consequence of biosynthetic enzyme abundance. IMPORTANCEPropionibacterium freudenreichii is a central bacterium used in food fermentations and one of the few microorganisms able to synthesize biologically active vitamin B12, making it valuable for industry and biotechnology. Yet the metabolic principles that govern its performance under different growth conditions remain poorly understood. Here, we show that carbon source is a key determinant of metabolic state, dictating how cells resolve redox constraints and allocate biosynthetic resources. We uncover a previously unrecognized adaptation in sugar-grown cells, where aspartate functions as an alternative electron sink to sustain redox balance under constrained conditions. By contrast, lactate supports a physiological state that promotes efficient vitamin B12 biosynthesis. These findings reveal a central role for carbon source in shaping metabolic configuration and identify redox balancing as a critical lever linking environmental inputs to biosynthetic output. More broadly, this work provides mechanistic insight into redox-constrained metabolism and a framework for improving vitamin B12 production and other microbial bioprocesses.

microbiology↗

bioRxiv2025

Proteomic characterization of extracellular vesicles from 12 commensal bacterial species

Bacterial extracellular vesicles (bEVs) produced by intestinal commensal bacteria mediate host-microbe interactions, but their proteomes have been explored in only a small number of species. We characterized bEVs from in vitro cultures of 12 species of Actinomycetota, Bacillota (Firmicutes), Bacteroidota, Fusobacteria, Pseudomonadota, and Verrucomicrobia. This is the first report of bEVs from Veillonella magna, an exceptional gram-negative Bacillota, and the gram-positives Peptostreptococcus russellii and Turicibacter sanguinis. The morphology and protein subcellular localization patterns of bEVs reflected the envelope structures of their parent bacteria. Notably, V. magna may be able to produce both outer membrane and cytoplasmic membrane vesicles. The proteome compositions were dictated by phylogeny, suggesting largely non-selective packaging of proteins. Annotation of protein functions indicated roles in nutrient metabolism and transport, and in host immune system modulation. Peptidoglycan modifying enzymes and the abundant bacteriophage proteins in Enterobacter cloacae and Limosilactobacillus reuteri bEVs may be involved in vesicle biogenesis. The functions of many abundant bEV proteins are unknown. The most abundant bEV proteins were largely species-specific, but we identified several conserved proteins that may be used as markers to distinguish commensal bEVs from host EVs. Comparison to previous in vitro and fecal metaproteomics data indicates that bEV proteome compositions are reproducible.

microbiology↗

bioRxiv2025

Intrinsic errors in mitochondrial translation trigger a decline in cell fitness.

Defects in the faithful expression of the human mitochondrial genome underlies disease states, from rare inherited disorders to common pathologies and the aging process itself. The ensuing decrease in the capacity for oxidative phosphorylation alone cannot account for the phenotype complexity associated with disease. Here, we address how aberrations in mitochondrial nascent chain synthesis per se exert a decline in cell fitness using a classic model of mitochondrial induced premature aging. We identify how intrinsic errors during mitochondrial nascent chain synthesis destabilize organelle gene expression, triggering intracellular stress responses that rewire cellular metabolism and cytokine secretion. Further, we show how these mechanisms extend to pathogenic variants associated with inherited human disorders. Together, our findings reveal how aberrations in mitochondrial protein synthesis can sensitize a cell to metabolic challenges associated with disease and pathogen infection independent of oxidative phosphorylation. Teaser/One-Sentence SummaryAberrations in mitochondrial translation elongation trigger activation of intracellular stress responses associated with disease and aging.

cell biology↗

bioRxiv2025

Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

T cells play a crucial role in the adaptive immune system and depend on tightly regulated intracellular signalling pathways to respond in an appropriate manner. Adapter proteins have flexible and dynamic features, which allow them to regulate T cell signal transduction pathways. As adapter proteins are enzymatically inert and may play multiple roles in parallel, it has been a challenge to fully characterise their functions individually. One such protein in T cells, is T cell specific adapter protein (TSAd), which is upregulated following T cell receptor (TCR) stimulation and is believed to mediate Src family tyrosine kinase signalling. However, the functional role remains elusive, possibly due to limited insight into interactors that potentially bind TSAd. The only structurally well-defined feature within TSAd, is the Src homology 2 (SH2) domain. This conserved domain displays prototypic binding of phosphorylated tyrosines, which suggests that the adapter molecule is implicated in phosphotyrosine signalling pathways. Here, we used an unbiased approach to identify ligands of the TSAd SH2 domain, by using affinity-purification mass spectrometry (AP-MS). Several novel ligands, many of which are known to be implicated in negative regulation of T cell intracellular signalling, were identified. More specifically, we showed that TSAd binds DOK2 and PTPN11 and determined the tyrosines responsible for the TSAd SH2 domain-dependent interaction. Ablation of TSAd and DOK2 by CRISPR/Cas9 in Jurkat T cells resulted in altered tyrosine phosphorylation. Taken together, these findings provide new insight into the possible function of TSAd as a negative signalling node in T cells.

immunology↗

bioRxiv2025

A bioinformatic workflow to facilitate the study of less-understood proteins: the case of SH2D2A

Adaptor proteins are key regulators of immune signalling but are challenging to study due to redundant pathways that obscure clear functional "anchor" phenotypes. Making use of public single-cell RNA sequencing (scRNA-seq) datasets, we theorised that anchors could be identified by working in the reverse direction: starting from the transcriptomic level and working "bottom-up" to identify anchoring phenomena at the cell surface. We have produced a prototype workflow for this method using the case of the lymphocyte-enriched adaptor protein SH2D2A, whose function remains uncertain. Using our bottom-up analysis, we have identified a link between SH2D2A and T cell-T cell (T-T) synapses enriched for ITGB2. The functional implications of this link are currently being explored, but further application of this bioinformatic approach to less-understood proteins may prove useful in understanding the minutiae of immune cell signalling, with implications for immunotherapy.

molecular biology↗

bioRxiv2024

Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases

The circulating antibody repertoire is crucial for immune protection, holding significant immunological and biotechnological value. While bottom-up mass spectrometry (MS) is the most widely used proteomics technique for profiling the sequence diversity of circulating antibodies (Ab-seq), it has not been thoroughly benchmarked. We quantified the replicability and robustness of Ab-seq using six monoclonal antibodies with known protein sequences in 70 different combinations of concentration and oligoclonality, both with and without polyclonal serum IgG background. Each combination underwent four protease treatments and was analyzed across four experimental and three technical replicates, totaling 3,360 LC-MS/MS runs. We quantified the dependence of MS-based Ab-seq identification on antibody sequence, concentration, protease, background signal diversity, and bioinformatics setups. Integrating the data from experimental replicates, proteases, and bioinformatics tools enhanced antibody identification. De novo peptide sequencing showed similar performance to database-dependent methods for higher antibody concentrations, but de novo antibody reconstruction remains challenging. Our work provides a foundational resource for the field of MS-based antibody profiling.

immunology↗

bioRxiv2024

Aerobic Adaptation and Metabolic Dynamics of Propionibacterium freudenreichii DSM 20271: Insights from Comparative Transcriptomics and Surfaceome Analysis

Propionibacterium freudenreichii (PFR) DSM 20271 is a bacterium known for its ability to thrive in diverse environments and to produce vitamin B12. Despite its anaerobic preference, recent studies have elucidated its ability to prosper in the presence of oxygen, prompting a deeper exploration of its physiology under aerobic conditions. Here, we investigated the response of DSM 20271 to aerobic growth by employing comparative transcriptomic and surfaceome analyses alongside metabolite profiling. Cultivation under controlled partial pressure of oxygen (pO2) conditions revealed significant increases in biomass formation and altered metabolite production, notably of B12 vitamin, pseudovitamin-B12, propionate and acetate, under aerobic conditions. Transcriptomic analysis identified differential expression of genes involved in lactate metabolism, TCA cycle, and electron transport chain, suggesting metabolic adjustments to aerobic environments. Moreover, surfaceome analysis unveiled growth environment-dependent changes in surface protein abundance, with implications for sensing and adaptation to atmospheric conditions. Supplementation experiments with key compounds highlighted the potential for enhancing aerobic growth, emphasizing the importance of iron and -ketoglutarate availability. Furthermore, in liquid culture, FeSO4 supplementation led to increased heme production and reduced vitamin B12 production, highlighting the impact of oxygen and iron availability on the metabolic pathways. These findings deepen our understanding of PFRs physiological responses to oxygen availability and offer insights for optimizing its growth in industrial applications. ImportanceThe study of the response of Propionibacterium freudenreichii to aerobic growth is crucial for understanding how this bacterium adapts to different environments and produces essential compounds like vitamin B12. By investigating its physiological changes under aerobic conditions, we can gain insights into its metabolic adjustments and potential for enhanced growth. These findings not only deepen our understanding of P. freudenreichii responses to oxygen availability but also offer valuable information for optimizing its growth in industrial applications. This research sheds light on the adaptive mechanisms of this bacterium, providing a foundation for further exploration and potential applications in various fields.

microbiology↗

bioRxiv2023

Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling

Immunoglobulins (Ig), which exist either as B-cell receptors (BCR) on the surface of B cells or as antibodies when secreted, play a key role in the recognition and response to antigenic threats. The capability to jointly characterize the BCR and antibody repertoire is crucial in understanding human adaptive immunity. From peripheral blood, bulk BCR sequencing (bulkBCR-seq) currently provides the highest sampling depth, single-cell BCR sequencing (scBCR-seq) allows for paired chain characterization, and antibody peptide sequencing by tandem mass spectrometry (Ab-seq) provides information on the composition of secreted antibodies in the serum. Although still rare, studies combining these three technologies would comprehensively capture the humoral immune response. Yet, it has not been benchmarked to what extent the datasets generated by these three technologies overlap and complement each other. To address this question, we isolated peripheral blood B cells from healthy donors and sequenced BCRs at bulk and single-cell level, in addition to utilizing publicly available sequencing data. Integrated analysis was performed on these datasets, resolved by replicates and across individuals. Simultaneously, serum antibodies were isolated, digested with multiple proteases, and analyzed with Ab-seq. Systems immunology analysis showed high concordance in repertoire features between bulk and scBCR-seq within individuals, especially when replicates were utilized. In addition, Ab-seq identified clonotype-specific peptides using both bulk and scBCR-seq library references, demonstrating the feasibility of combining scBCR-seq and Ab-seq for reconstructing paired-chain Ig sequences from the serum antibody repertoire. Collectively, our work serves as a proof-of-principle for combining bulk sequencing, single-cell sequencing, and mass spectrometry as complementary methods towards capturing humoral immunity in its entirety.

immunology↗

bioRxiv2022

Towards optimised extracellular vesicle proteomics from cerebrospinal fluid

The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean {+/-} standard deviation. Using proteomics, SEC fractions 3-5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4-5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6ml, 3ml, 1ml, and 0.5ml) to evaluate the effect on the proteomic profile. Even with a 0.5ml starting volume, 743{+/-}77 or 345{+/-}88 proteins were identified depending on whether matches between runs was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5ml of canine CSF.

cell biology↗

bioRxiv2022

Supernumerary proteins of the human mitochondrial ribosomal small subunit are integral for assembly and translation

Mitochondrial ribosomes (mitoribosomes) have undergone substantial structural remodelling throughout evolution. Compared to their prokaryotic counterparts, mitoribosomes show a substantial loss of ribosomal RNA, whilst acquiring unique protein subunits located on the periphery of the ribosomal subunit structures. We set out to investigate the functional properties of all 14 unique (mitochondrial-specific or supernumerary) human mitoribosomal proteins in the small subunit. Using genome editing with CRISPR-Cas9, we made knockouts for each subunit in HEK293 cells to study the effect on mitoribosome assembly and function in protein synthesis. Unexpectedly, we show that each supernumerary knockout leads to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Our data demonstrates that all supernumerary subunits are essential structural components except mS37. Surprisingly, we found the stability of mS37 was reduced in all our supernumerary knockouts of the small and large ribosomal subunits as well as patient-derived lines with mitoribosome assembly defects. We identified that a redox regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.

molecular biology↗

bioRxiv2021

Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance and Persistence

The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM-proteins of Mycobacterium marinum ATCC927, a non-tuberculous mycobacterial model, was monitored over three months by label-free proteomics and compared with cell-surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC927 forms pellicle-type (PBFs) and submerged-type (SBFs) biofilms after two weeks and two days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggests that specific changes in mycolic acid synthesis (cord factor), Esx1-secretion, and cell-wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting the 64 x MIC rifampicin (persisters) were detected in both biofilm subtypes, and already in one-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of varying ambient oxygen tensions on each cell type and non-classical protein secretion are likely factors explaining majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in PL and PBF cells, while SBF may prefer both the Esx5- and non-classical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports a first proteomic insight into aging mycobacterial biofilm-ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence on non-tuberculous mycobacteria. IMPORTANCEMycobacteria are naturally resilient and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of TB. The present study shows that non-tuberculous Mycobacterium marinum ATCC927 forms submerged- and pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance- and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.

microbiology↗

bioRxiv2020

Scalable production of tissue-like vascularised liver organoids from human PSCs.

A lack of physiological parity between 2D cell culture and in vivo, has paved the way towards more organotypic models. Organoids exist for a number of tissues, including the liver. However, current approaches to generate hepatic organoids suffer drawbacks, including a reliance on extracellular matrices (ECM), the requirement to pattern in 2D culture, costly growth factors and a lack of cellular diversity, structure and organisation. Current hepatic organoid models are generally simplistic, composed of hepatocytes or cholangiocytes, which renders them less physiologically relevant when compared to native tissue. Here we aim to address these drawbacks. To address this, we have developed an approach that does not require 2D patterning, is ECM independent combined with small molecules to mimic embryonic liver development that produces massive quantities of liver like organoids. Using single-cell RNA sequencing and immunofluorescence we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, innervation and a population of resident macrophage - the Kupffer cells. The organoids exhibit key liver functions including drug metabolism, serum protein production, coagulation factor production, bilirubin uptake and urea synthesis. The organoids can be transplanted and maintained in mice producing human albumin long term. The organoids exhibit a complex cellular repertoire reflective of the organ, have de novo vascularization and innervation, enhanced function and maturity. This is a pre-requisite for a myriad of applications from cellular therapy, tissue engineering, drug toxicity assessment, disease modeling, to basic developmental biology.

cell biology↗

bioRxiv2019

Growth Mode and Carbon Source Impact the Surfaceome Dynamics of Lactobacillus rhamnosus GG

Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC/A pilus and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN and PepA). The abundance of several known adhesins and novel moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The classical surface adhesins were predicted to be more abundant on planktonic cells growing either on fructose (MBF and SpaA) or glucose (SpaC). An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate classical and nonclassical protein export in GG, which ensures the presence of an integral and coordinated system that contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.

microbiology↗