Journal: Life Science Alliance

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EMBO Press

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2575-1077

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2018 - 201912020 - 202510
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Publications 1 - 10 of 11
  • Acute and chronic effects of a light-activated FGF receptor in keratinocytes in vitro and in mice
    Item type: Journal Article
    Rauschendorfer, Theresa; Gurri, Selina; Heggli, Irina; et al. (2021)
    Life Science Alliance
    FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with re-markable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 acti-vation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of re-sponsiveness to light activation. These results demonstrate un-expected cell type-specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.
  • Multi-region proteome analysis quantifies spatial heterogeneity of prostate tissue biomarkers
    Item type: Journal Article
    Guo, Tiannan; Li, Li; Zhong, Qing; et al. (2018)
    Life Science Alliance
    It remains unclear to what extent tumor heterogeneity impacts on protein biomarker discovery. Here, we quantified proteome intra-tissue heterogeneity (ITH) based on a multi-region analysis of prostate tissues using pressure cycling technology and Sequential Windowed Acquisition of all THeoretical fragment ion mass spectrometry. We quantified 6,873 proteins and analyzed the ITH of 3,700 proteins. The level of ITH varied depending on proteins and tissue types. Benign tissues exhibited more complex ITH patterns than malignant tissues. Spatial variability of 10 prostate biomarkers was validated by immunohistochemistry in an independent cohort (n = 83) using tissue microarrays. Prostate-specific antigen was preferentially variable in benign prostatic hyperplasia, whereas growth/differentiation factor 15 substantially varied in prostate adenocarcinomas. Furthermore, we found that DNA repair pathways exhibited a high degree of variability in tumorous tissues, which may contribute to the genetic heterogeneity of tumors. This study conceptually adds a new perspective to protein biomarker discovery: it suggests that recent technological progress should be exploited to quantify and account for spatial proteome variation to complement biomarker identification and utilization.
  • VE-cadherin junction dynamics in initial lymphatic vessels promotes lymph node metastasis
    Item type: Journal Article
    Sáinz-Jaspeado, Miguel; Ring, Sarah; Proulx, Steven T.; et al. (2024)
    Life Science Alliance
    The endothelial junction component vascular endothelial (VE)-cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2Y949F/Y949F ) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.
  • Conserved exchange of paralog proteins during neuronal differentiation
    Item type: Journal Article
    Di Fraia, Domenico; Anitei, Mihaela; Mackmull, Marie-Therese; et al. (2022)
    Life Science Alliance
    Gene duplication enables the emergence of new functions by lowering the evolutionary pressure that is posed on the ancestral genes. Previous studies have highlighted the role of specific paralog genes during cell differentiation, for example, in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. Whereas ∼80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B members of the COPII complex. Altering the ratio between these two genes via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.
  • TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44
    Item type: Journal Article
    Soldà, Tatiana; Galli, Carmela; Guerra, Concetta; et al. (2024)
    Life Science Alliance
    The ER is the organelle of nucleated cells that produces lipids, sugars, and proteins. More than 20 ER-resident members of the protein disulfide isomerase (PDI) family regulate formation, isomerization, and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4, and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least-known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multipass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57, and ERp44. The association between TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER localization of TMX5 in pre-Golgi compartments. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1β, Prx4, ERAP1, and SUMF1 that operate in pre-Golgi compartments but lack localization sequences required to position themselves and rely on ERp44 engagement for proper intercompartmental distribution.
  • The hGIDᴳᴵᴰ⁴ E3 ubiquitin ligase complex targets ARHGAP11A to regulate cell migration
    Item type: Journal Article
    Bagci, Halil; Winkler, Martin; Grädel, Benjamin; et al. (2024)
    Life Science Alliance
    The human CTLH/GID (hGID) complex emerged as an important E3 ligase regulating multiple cellular processes, including cell cycle progression and metabolism. However, the range of biological functions controlled by hGID remains unexplored. Here, we used proximity-dependent biotinylation (BioID2) to identify proteins interacting with the hGID complex, among them, substrate candidates that bind GID4 in a pocket-dependent manner. Biochemical and cellular assays revealed that the hGIDᴳᴵᴰ⁴ E3 ligase binds and ubiquitinates ARHGAP11A, thereby targeting this RhoGAP for proteasomal degradation. Indeed, GID4 depletion or impeding the GID4 substrate binding pocket with the PFI-7 inhibitor stabilizes ARHGAP11A protein amounts, although it carries no functional N-terminal degron. Interestingly, GID4 inactivation impairs cell motility and directed cell movement by increasing ARHGAP11A levels at the cell periphery, where it inactivates RhoA. Together, we identified a wide range of hGIDᴳᴵᴰ⁴ E3 ligase substrates and uncovered a unique function of the hGIDᴳᴵᴰ⁴ E3 ligase regulating cell migration by targeting ARHGAP11A.
  • Convergent evolution and B-cell recirculation in germinal centers in a human lymph node
    Item type: Journal Article
    Pelissier, Aurelien; Stratigopoulou, Maria; Donner, Naomi; et al. (2023)
    Life Science Alliance
    Germinal centers (GCs) play a central role in generating an effective immune response against infectious pathogens, and failures in their regulating mechanisms can lead to the development of autoimmune diseases and cancer. Although previous works study experimental systems of the immune response with mouse models that are immunized with specific antigens, our study focused on a real-life situation, with an ongoing GC response in a human lymph node (LN) involving multiple asynchronized GCs reacting simultaneously to unknown antigens. We combined laser capture microdissection of individual GCs from human LN with next-generation repertoire sequencing to characterize individual GCs as distinct evolutionary spaces. In line with well-characterized GC responses in mice, elicited by immunization with model antigens, we observe a heterogeneous clonal diversity across individual GCs from the same human LN. Still, we identify shared clones in several individual GCs, and phylogenetic tree analysis combined with paratope modeling suggest the re-engagement and rediversification of B-cell clones across GCs and expanded clones exhibiting shared antigen responses across distinct GCs, indicating convergent evolution of the GCs.
  • Proteomic-based stratification of intermediate-risk prostate cancer patients
    Item type: Journal Article
    Zhong, Qing; Sun, Rui; Aref, Adel T.; et al. (2024)
    Life Science Alliance
    Gleason grading is an important prognostic indicator for prostate adenocarcinoma and is crucial for patient treatment decisions. However, intermediate-risk patients diagnosed in the Gleason grade group (GG) 2 and GG3 can harbour either aggressive or non-aggressive disease, resulting in under- or overtreatment of a significant number of patients. Here, we performed proteomic, differential expression, machine learning, and survival analyses for 1,348 matched tumour and benign sample runs from 278 patients. Three proteins (F5, TMEM126B, and EARS2) were identified as candidate biomarkers in patients with biochemical recurrence. Multivariate Cox regression yielded 18 proteins, from which a risk score was constructed to dichotomize prostate cancer patients into low- and high-risk groups. This 18-protein signature is prognostic for the risk of biochemical recurrence and completely independent of the intermediate GG. Our results suggest that markers generated by computational proteomic profiling have the potential for clinical applications including integration into prostate cancer management.
  • BACH1 as a key driver in rheumatoid arthritis fibroblast-like synoviocytes identified through gene network analysis
    Item type: Journal Article
    Pelissier, Aurelien; Laragione, Teresina; Harris, Carolyn; et al. (2025)
    Life Science Alliance
    RNA-sequencing and differential gene expression studies have significantly advanced our understanding of pathogenic pathways underlying rheumatoid arthritis (RA). Yet, little is known about cell-specific regulatory networks and their contributions to disease. In this study, we focused on fibroblast-like synoviocytes (FLS), a cell type central to disease pathogenesis and joint damage in RA. We used a strategy that computed sample-specific gene regulatory networks to compare network properties between RA and osteoarthritis FLS. We identified 28 transcription factors (TFs) as key regulators central to the signatures of RA FLS. Six of these TFs are new and have not been previously implicated in RA through ex vivo or in vivo studies, and included BACH1, HLX, and TGIF1. Several of these TFs were found to be co-regulated, and BACH1 emerged as the most significant TF and regulator. The main BACH1 targets included those implicated in fatty acid metabolism and ferroptosis. The discovery of BACH1 was validated in experiments with RA FLS. Knockdown of BACH1 in RA FLS significantly affected the gene expression signatures, reduced cell adhesion and mobility, interfered with the formation of thick actin fibers, and prevented the polarized formation of lamellipodia, all required for the RA destructive behavior of FLS. This study establishes BACH1 as a central regulator of RA FLS phenotypes and suggests its potential as a therapeutic target to selectively modulate RA FLS.
  • ATP-independent substrate recruitment to proteasomal degradation in mycobacteria
    Item type: Journal Article
    von Rosen, Tatjana; Pepelnjak, Monika; Quast, Jean-Philipp; et al. (2023)
    Life Science Alliance
    Mycobacteria and other actinobacteria possess proteasomal degradation pathways in addition to the common bacterial compartmentalizing protease systems. Proteasomal degradation plays a crucial role in the survival of these bacteria in adverse environments. The mycobacterial proteasome interacts with several ring-shaped activators, including the bacterial proteasome activator (Bpa), which enables energy-independent degradation of heat shock repressor HspR. However, the mechanism of substrate selection and processing by the Bpa-proteasome complex remains unclear. In this study, we present evidence that disorder in substrates is required but not sufficient for recruitment to Bpa-mediated proteasomal degradation. We demonstrate that Bpa binds to the folded N-terminal helix-turn-helix domain of HspR, whereas the unstructured C-terminal tail of the substrate acts as a sequence-specific threading handle to promote efficient proteasomal degradation. In addition, we establish that the heat shock chaperone DnaK, which interacts with and co-regulates HspR, stabilizes HspR against Bpa-mediated proteasomal degradation. By phenotypical characterization of Mycobacterium smegmatis parent and bpa deletion mutant strains, we show that Bpa-dependent proteasomal degradation supports the survival of the bacterium under stress conditions by degrading HspR that regulates vital chaperones.
Publications 1 - 10 of 11