Biology subjects

Fijalkowska, D.

Publications and source records attributed to Fijalkowska, D..

13 recordsLinked to original sources

bioRxiv2025

Optimizing efficacy to safety ratio of glucocorticoids in rheumatoid arthritis models by leveraging PPARα agonism

ObjectivesGlucocorticoids remain essential therapies for several immune- and inflammatory diseases such as rheumatoid arthritis (RA) but are notorious for their (metabolic) side effects. Given the anti-inflammatory and metabolically favorable actions of peroxisome-proliferator activated nuclear receptor (PPAR) agonists, we investigated whether PPAR agonism could enhance the therapeutic efficacy and/or mitigate the (metabolic) side effects of glucocorticoids. MethodsWe evaluated the effects of the synthetic glucocorticoid dexamethasone and the PPAR agonist GW7647 (GW) across three RA model systems: L929sA fibroblasts, primary human fibroblast-like synoviocytes (FLS) and collagen-induced arthritis (CIA) mice. ResultsDexamethasone reduced the inflammatory TNF response in L929sA cells, which was further potentiated by GW. In vivo however, GW reduced the dexamethasone-induced adiposity and hypertriglyceridemia, but not arthritis severity. Curiously, GW alone induced several proinflammatory genes within arthritic synovium which were counteracted by glucocorticoids. Proteomic profiling of TNF-stimulated human FLS revealed that combined use of dexamethasone and GW selectively suppressed interferon-stimulated proteins. In line herewith, co-stimulation with TNF and IFN{beta} amplified the suppressive effect of combined dexamethasone and GW treatment on pro-inflammatory gene expression in L929sA versus TNF alone. ConclusionGW enhances the anti-inflammatory effects of glucocorticoids in human FLS and L929sA, and mitigates metabolic side effects of dexamethasone in vivo, without compromising their efficacy. In addition, PPAR agonism permits to broaden its anti-inflammatory profile to interferon driven pathways. Given that both synthetic glucocorticoids and PPAR agonists are already widely used in (general) clinical practice, these findings offer a promising strategy to optimize glucocorticoid-based therapies. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSO_LISynthetic glucocorticoids such as dexamethasone are widely used as immunosuppressive drugs, but cause many (metabolic) side effects. C_LIO_LIPeroxisome-proliferator-activated nuclear receptor (PPAR) agonists are clinically primarily used to treat symptoms that resemble glucocorticoid-induced side effects, but they also exert (modest) immunosuppressive effects. C_LI What this study addsO_LIThis study explores the therapeutic potential of a combination treatment with dexamethasone and PPAR agonist GW7647 (GW) in cellular and murine models of rheumatoid arthritis C_LIO_LIWe reveal that GW attenuates dexamethasone-induced adiposity and hypertriglyceridemia in vivo, hereby improving glucocorticoid-related side effects. C_LIO_LIThe therapeutic efficacy of dexamethasone is maintained or even enhanced by GW in murine and cellular models of rheumatic arthritis, respectively. C_LIO_LIWe offer novel mechanistic insights in the proposed combination treatment by revealing the selective suppression of interferon signaling pathways in human FLS. C_LI How this study might affect research, practice or policyO_LIGiven that both synthetic glucocorticoids and PPAR agonists are already used in clinical practice, this study offers a promising, translatable strategy to optimize glucocorticoid-based therapies with an improved efficacy/safety ratio. C_LI

immunology↗

bioRxiv2025

Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)- based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.

genomics↗

bioRxiv2024

PPARalpha-ERRalpha crosstalk mitigates metabolic dysfunction-associated steatotic liver disease progression

Background and aimsMetabolic dysfunction-associated steatotic liver disease (MASLD) is now the most common liver disease worldwide. This study investigates how targeting two key nuclear receptors involved in hepatic energy metabolism, peroxisome proliferator-activated receptor alpha (PPAR) and estrogen-related receptor alpha (ERR), impacts MASLD. MethodsThe PPAR agonist pemafibrate and/or ERR inverse agonist C29 were administered in a short- and long-term Western diet plus fructose model, and a diabetic-background streptozotocin-Western diet model (STZ-WD). Liver morphology, histological samples, serum metabolites, RNA and protein levels were analysed and scanning electron microscopy was performed. In addition, we performed cell-based assays and immunohistochemistry and immunofluorescence stainings with light and super-resolution confocal microscopy of healthy, MASLD and MASH human livers. ResultsThe ligand combinations efficacy was underscored by reduced liver steatosis in all the mouse models. Both long-term models showed improvements in body weight and liver morphology, alongside reductions in inflammation and fibrosis. Additionally, tumor formation was prevented in the STZ-WD mice model. Cell-based assays demonstrated that ERR inhibits PPARs activity, likely explaining why ERR blockage improves inflammatory and lipid metabolism gene profiles and enhances lipid-lowering effects. Complementary RNA sequencing and shotgun proteomics, combined with enrichment analysis, jointly identified downregulated serum amyloid A1/A2 as an essential component underlying the combination treatments effectiveness. MASLD/MASH patient livers showed reduced PPAR and increased ERR levels supporting disrupted NR crosstalk in the hepatocyte nucleus. ConclusionOur study comprehensively supports that dual nuclear receptor targeting by simultaneously increasing PPAR and diminishing ERR activity may represent a viable novel strategy against MASLD. Lay summaryOur study using three distinct MASLD mouse models shows that a simultaneous targeting of PPAR and ERR reduces liver fat, fibrosis, inflammation, and tumor formation. The combination treatment modifies lipid metabolism pathways, and uniquely lowers levels of serum amyloid A1/A2 in the liver. Impact and implicationsOur research introduces a novel therapeutic strategy against MASLD by simultaneously increasing PPAR activity while diminishing ERR activity. With PPAR agonists already tested in phase III clinical trials, ERR ligands/modulators need further (clinical) development to make our findings applicable to both MASLD patients and physicians.

pharmacology and toxicology↗

bioRxiv2024

Yeast-based production platform for potent and stable heavy chain-only antibodies

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.

microbiology↗

bioRxiv2024

Crosstalk interactions between transcription factors ERRalpha and PPARalpha assist PPARalpha-mediated gene expression

The peroxisome proliferator-activated receptor (PPAR) is a crucial transcription factor governing genes associated with fatty acid {beta}-oxidation. How various interacting proteins modulate PPARs transcriptional function remains incompletely understood. Employing an unbiased mammalian protein-protein interaction trap with liganded PPAR as bait, we identified an interaction with the orphan nuclear receptor estrogen-related receptor (ERR). Random mutagenesis scanning of PPARs ligand-binding domain and coregulator profiling experiments implicated bridging coregulators, while in vitro studies suggested a trimeric interaction involving RXR. The PPAR{middle dot}ERR interaction, dependent on three C-terminal residues within ERRs helix 12, was reinforced by PGC1 and serum deprivation. Pharmacological inhibition of ERR reduced its interaction with ligand-activated PPAR, revealing a transcriptome indicative of ERR functioning as a transcriptional repressor on prototypical PPAR target genes. Intriguingly, ERR exhibited opposite behavior on other PPAR targets, including the isolated PDK4 enhancer. Chromatin immunoprecipitation analyses demonstrated PPAR ligand-dependent recruitment of ERR onto specific chromatin regions where PPAR binds in mouse livers. These findings highlight intricate transcriptional crosstalk mechanisms between PPAR and ERR, suggesting a multi-layered regulatory network fine-tuning PPARs activity as a nutrient-sensing transcription factor.

molecular biology↗

bioRxiv2023

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods, and has quickly become the current state-of-the-art in the field. Nevertheless, tight control of proximity labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein-of-interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split-link design, we applied it to identify protein interactions of the glucocorticoid receptor and SARS-CoV-2 nucleocapsid and NSP7 proteins by TurboID proximity labeling. Our results demonstrate that our T2A split-link provides an opportune control that builds upon previously established control requirements in the field. MotivationIn proximity labeling proteomics protein-protein interactions are identified by in vivo biotinylation. However, the current lack of a universally applicable negative control for differential analysis affects accurate mapping of the interactome. To bridge this gap, we conceptualized a system based on the T2A self-cleaving peptide to match expression levels between control and bait protein setups while using the same bait protein. In addition, we implemented a versatile modular cloning system to build mammalian expression vectors for, but not limited to, proximity labeling assays.

biochemistry↗

bioRxiv2023

Crosstalk between the glucocorticoid and mineralocorticoid receptor boosts glucocorticoid-induced killing of multiple myeloma cells

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that culminates in improved myeloma cell killing. We show that the GR agonist Dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist Spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR-MR homodimerization, while leaving GR-GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

cancer biology↗

bioRxiv2023

Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that bind a conserved membrane proximal epitope of the spike

Currently circulating SARS-CoV-2 variants have gained complete or significant resistance to all SARS-CoV-2-neutralizing antibodies that have been used in the clinic. Such antibodies can prevent severe disease in SARS-CoV-2 exposed patients for whom vaccines may not provide optimal protection. Here, we describe single-domain antibodies (VHHs), also known as nanobodies, that can broadly neutralize SARS-CoV-2 with unusually high potency. Structural analysis revealed their binding to a unique, highly conserved, membrane proximal, quaternary epitope in the S2 subunit of the spike. Furthermore, a VHH-human IgG1 Fc fusion, efficiently expressed in Chinese hamster ovary cells as a stable antibody construct, protected hamsters against SARS-CoV-2 replication in a therapeutic setting when administered systemically at low dose. This VHH-based antibody represents a new candidate anti-COVID-19 biologic that targets the Achilles heel of the viral spike.

microbiology↗

bioRxiv2023

N-terminal proteoforms may engage in different protein complexes

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared to their canonical counterparts. Such proteoforms can have altered localizations, stabilities and functions. While proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. Additionally, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared to their canonical counterpart, thus further expanding the functional diversity of proteomes.

systems biology↗

bioRxiv2022

OPENPichia: building a free-to-operate Komagataella phaffii protein expression toolkit

In the standard toolkit for recombinant protein expression, the yeast known in biotechnology as Pichia pastoris (formally: Komagataella phaffii) takes up the position between E. coli and HEK293 or CHO mammalian cells, and is used by thousands of laboratories both in academia and industry. The organism is eukaryotic yet microbial, and grows to extremely high cell densities while secreting proteins into its fully defined growth medium, using very well established strong inducible or constitutive promoters. Many products made in Pichia are in the clinic and in industrial markets. Pichia is also a favoured host for the rapidly emerging area of precision fermentation for the manufacturing of food proteins. However, the earliest steps in the development of the industrial strain (NRRL Y-11430/CBS 7435) that is used throughout the world were performed prior to 1985 in industry (Phillips Petroleum Company) and are not in the public domain. Moreover, despite the long expiry of associated patents, the patent deposit NRRL Y-11430/CBS 7435 that is the parent to all commonly used industrial strains, is not or no longer made freely available through the resp. culture collections. This situation is far from ideal for what is a major chassis for synthetic biology, as it generates concern that novel applications of the system are still encumbered by licensing requirements of the very basic strains. In the spirit of open science and freedom to operate for what is a key component of biotechnology, we set out to resolve this by using genome sequencing of type strains, reverse engineering where necessary, and comparative protein expression and strain characterisation studies. We find that the industrial strains derive from the K. phaffii type strain lineage deposited as 54-11.239 in the UC Davis Phaff Yeast Strain collection by Herman Phaff in 1954. This type strain has valid equivalent deposits that are replicated/derived from it in other yeast strain collections, incl. in ARS-NRRL NRRL YB-4290 (deposit also made by Herman Phaff) and NRRL Y-7556, CBS 2612 and NCYC 2543. We furthermore discovered that NRRL Y-11430 and its derivatives carry an ORF-truncating mutation in the HOC1 cell wall synthesis gene, and that reverse engineering of a similar mutation in the NCYC 2543 type strain imparts the high transformability that is characteristic of the industrial strains. Uniquely, the NCYC 2543 type strain, which we propose to call OPENPichia henceforth, is freely available from the NCYC culture collection, incl. resale and commercial production licenses at nominal annual licensing fees1. Furthermore, our not-for-profit research institute VIB has also acquired a resale/distribution license from NCYC, which we presently use to openly provide to end-users our genome-sequenced OPENPichia subclone strain and its derivatives, i.e., currently the highly transformable hoc1tr and the his4 auxotrophic mutants. To complement the OPENPichia platform, a fully synthetic modular gene expression vector building toolkit was developed, which is also openly distributed, for any purpose. We invite other researchers to contribute to our open science resource-building effort to establish a new unencumbered standard chassis for Pichia synthetic biology.

molecular biology↗

bioRxiv2021

Limited, but potentially functional translation of non-coding transcripts in the HEK293T cellular cytosol

Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins, while other studies failed to find evidence for such types of non-canonical translation products. Here, we attempted to discover translation products from non-coding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. Theoretically, we show that our strategy facilitates the detection of translation events of transcripts from non-coding regions, but experimentally only find 19 peptides (less than 1% of all identified peptides) that might originate from such translation events. Virotrap based interactome analysis of two N-terminal proteoforms originating from non-coding regions finally showed the functional potential of these novel proteins.

molecular biology↗

bioRxiv2021

Drug development of an affinity enhanced, broadly neutralizing heavy chain only antibody that restricts SARS-CoV-2 in hamsters

We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

molecular biology↗

bioRxiv2020

Single cell 3'UTR analysis identifies changes in alternative polyadenylation throughout neuronal differentiation and in autism

Autism spectrum disorder (ASD) is a neurodevelopmental disease affecting social behavior. Many of the high-confident ASD risk genes relate to mRNA translation. Specifically, many of these genes are involved in regulation of gene expression for subcellular compartmentalization of proteins1. Cis-regulatory motifs that often localize to 3- and 5-untranslated regions (UTRs) offer an additional path for posttranscriptional control of gene expression. Alternative cleavage and polyadenylation (APA) affect 3UTR length thereby influencing the presence or absence of regulatory elements. However, APA has not yet been addressed in the context of neurodevelopmental disorders. Here we used single cell 3end sequencing to examine changes in 3UTRs along the differentiation from neural stem cells (NSCs) to neuroblasts within the adult brain. We identified many APA events in genes involved in neurodevelopment, many of them being high confidence ASD risk genes. Further, analysis of 3UTR lengths in single cells from ASD and healthy individuals detected longer 3UTRs in ASD patients. Motif analysis of modulated 3UTRs in the mouse adult neurogenic lineage and ASD-patients revealed enrichment of the cytoplasmic and polyadenylation element (CPE). This motif is bound by CPE binding protein 4 (CPEB4). In human and mouse data sets we observed co-regulation of CPEB4 and the CPEB-binding synaptic adhesion molecule amyloid beta precursor-like protein 1 (APLP1). We show that mice deficient in APLP1 show aberrant regulation of APA, decreased number of neural stem cells, and autistic-like traits. Our findings indicate that APA is used for control of gene expression along neuronal differentiation and is altered in ASD patients.

neuroscience↗