| 1. |
- Abraham, Elena T., et al.
(författare)
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Collagen's primary structure determines collagen:HSP47 complex stoichiometry
- 2021
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258. ; 297:6
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Tidskriftsartikel (refereegranskat)abstract
- Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.
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| 2. |
- Adamo, Christin S., et al.
(författare)
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EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis
- 2022
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Ingår i: American Journal of Human Genetics. - : Elsevier BV. - 0002-9297. ; 109:12, s. 2230-2252
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Tidskriftsartikel (refereegranskat)abstract
- EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.
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| 3. |
- Dill, Veronika, et al.
(författare)
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Biological dermal templates with native collagen scaffolds provide guiding ridges for invading cells and may promote structured dermal wound healing
- 2020
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Ingår i: International Wound Journal. - : Wiley. - 1742-4801 .- 1742-481X. ; 17:3, s. 618-630
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Tidskriftsartikel (refereegranskat)abstract
- Dermal substitutes are of major importance in treating full thickness skin defects. They come in a variety of materials manufactured into various forms, such as films, hydrocolloids, hydrogels, sponges, membranes, and electrospun micro- and nanofibers. Bioactive dermal substitutes act in wound healing either by delivery of bioactive compounds or by being constructed from materials having endogenous activity. The healing success rate is highly determined by cellular and physiological processes at the host-biomaterial interface during crucial wound healing steps. Hence, it is important to design appropriate wound treatment strategies with the ability to work actively with tissues and cells to enhance healing. Therefore, in this study, we investigated biological dermal templates and their potential to stimulate natural cell adherence, guidance, and morphology. The most pronounced effect was observed in biomaterials with the highest content of native collagen networks. Cell attachment and proliferation were significantly enhanced on native collagen scaffolds. Cell morphology was more asymmetrical on such scaffolds, resembling native in vivo structures. Importantly, considerably lower expression of myofibroblast phenotype was observed on native collagen scaffolds. Our data suggest that this treatment strategy might be beneficial for the wound environment, with the potential to promote improved tissue regeneration and reduce abnormal scar formation.
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| 4. |
- Gialeli, Chrysostomi, et al.
(författare)
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Complement inhibitor CSMD1 modulates epidermal growth factor receptor oncogenic signaling and sensitizes breast cancer cells to chemotherapy
- 2021
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Ingår i: Journal of Experimental and Clinical Cancer Research. - : Springer Science and Business Media LLC. - 1756-9966. ; 40:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Human CUB and Sushi multiple domains 1 (CSMD1) is a large membrane-bound tumor suppressor in breast cancer. The current study aimed to elucidate the molecular mechanism underlying the effect of CSMD1 in highly invasive triple negative breast cancer (TNBC). Methods: We examined the antitumor action of CSMD1 in three TNBC cell lines overexpressing CSMD1, MDA-MB-231, BT-20 and MDA-MB-486, in vitro using scanning electron microscopy, proteome array, qRT-PCR, immunoblotting, proximity ligation assay, ELISA, co-immunoprecipitation, immunofluorescence, tumorsphere formation assays and flow cytometric analysis. The mRNA expression pattern and clinical relevance of CSMD1 were evaluated in 3520 breast cancers from a modern population-based cohort. Results: CSMD1-expressing cells had distinct morphology, with reduced deposition of extracellular matrix components. We found altered expression of several cancer-related molecules, as well as diminished expression of signaling receptors including Epidermal Growth Factor Receptor (EGFR), in CSMD1-expressing cells compared to control cells. A direct interaction of CSMD1 and EGFR was identified, with the EGF-EGFR induced signaling cascade impeded in the presence of CSMD1. Accordingly, we detected increased ubiquitination levels of EGFR upon activation in CSMD1-expressing cells, as well as increased degradation kinetics and chemosensitivity. Accordingly, CSMD1 expression rendered tumorspheres pretreated with gefitinib more sensitive to chemotherapy. In addition, higher mRNA levels of CSMD1 tend to be associated with better outcome of triple negative breast cancer patients treated with chemotherapy. Conclusions: Our results indicate that CSMD1 cross-talks with the EGFR endosomal trafficking cascade in a way that renders highly invasive breast cancer cells sensitive to chemotherapy. Our study unravels one possible underlying molecular mechanism of CSMD1 tumor suppressor function and may provide novel avenues for design of better treatment.
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| 5. |
- Köhler, Anna, et al.
(författare)
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New specific HSP47 functions in collagen subfamily chaperoning
- 2020
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Ingår i: FASEB Journal. - 0892-6638. ; 34:9, s. 12040-12052
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Tidskriftsartikel (refereegranskat)abstract
- Although collagens are the most abundant proteins implicated in various disease pathways, essential mechanisms required for their proper folding and assembly are poorly understood. Heat-shock protein 47 (HSP47), an ER-resident chaperone, was mainly reported to fulfill key functions in folding and secretion of fibrillar collagens by stabilizing pro-collagen triple-helices. In this study, we demonstrate unique functions of HSP47 for different collagen subfamilies. Our results show that HSP47 binds to the N-terminal region of procollagen I and is essential for its secretion. However, HSP47 ablation does not majorly impact collagen VI secretion, but its lateral assembly. Moreover, specific ablation of Hsp47 in murine keratinocytes revealed a new role for the transmembrane collagen XVII triple-helix formation. Incompletely folded collagen XVII C-termini protruding from isolated HSP47 null keratinocyte membrane vesicles could be fully restored upon the application of recombinant HSP47. Thus, our study expands the current view regarding the client repertoire and function of HSP47, as well as emphasizes its importance for transmembrane collagen folding.
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| 6. |
- Linders, Johan, et al.
(författare)
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Complement Component 3 Is Required for Tissue Damage, Neutrophil Infiltration, and Ensuring NET Formation in Acute Pancreatitis
- 2020
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Ingår i: European Surgical Research. - : S. Karger AG. - 0014-312X .- 1421-9921. ; 61:6, s. 163-176
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Neutrophil extracellular traps (NETs) are known to play an important role in the pathophysiology of acute pancreatitis (AP). Activation of the complement cascade has been shown to occur in AP. The aim of this study was to examine whether complement component 3 is involved in the generation of NETs in AP.METHODS: AP was induced in wild-type and C3-deficient mice by retrograde infusion of taurocholate into the pancreatic duct. Blood, lung, and pancreas tissue were collected and MPO activity was determined in lung and pancreas tissue. Histological examination of the inflamed pancreas was performed. Plasma levels of CXCL2, MMP-9, IL-6, and DNA-histone complexes as well as pancreatic levels of CXCL1 and CXCL2 were determined by use of enzyme-linked immunosorbent assay. NETs were detected in the pancreas by electron microscopy. The amount of MPO and citrullinated histone 3 in neutrophils isolated from bone marrow was examined using flow cytometry.RESULTS: In C3-deficient mice, challenge with taurocholate yielded much fewer NETs in the pancreatic tissue compared with wild-type controls. Taurocholate-induced blood levels of amylase, tissue injury, and neutrophil recruitment in the pancreas were markedly reduced in the mice lacking C3. Furthermore, MPO levels in the lung, and plasma levels of IL-6, MMP-9, and CXCL2 were significantly lower in the C3-deficient mice compared to wild-type mice after the induction of AP. In vitro studies revealed that neutrophils from C3-deficient mice had normal NET-forming ability and recombinant C3a was not capable of directly inducing NETs formation in the wild-type neutrophils.CONCLUSION: C3 plays an important role in the pathophysiology of AP as it is necessary for the recruitment of neutrophils into the pancreas and ensuring NETs formation. Targeting C3 could hence be a potential strategy to ameliorate local damage as well as remote organ dysfunction in AP.
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| 7. |
- Linders, Johan, et al.
(författare)
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Extracellular cold-inducible RNA-binding protein regulates neutrophil extracellular trap formation and tissue damage in acute pancreatitis
- 2020
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Ingår i: Laboratory Investigation. - : Elsevier BV. - 0023-6837. ; 100:12, s. 1618-1630
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophil extracellular traps (NETs) play a key role in the development of acute pancreatitis (AP). In the present study, we studied the role of extracellular cold-inducible RNA-binding protein (eCIRP), a novel damage-associated-molecular-pattern molecule, in severe AP. C57BL/6 mice underwent retrograde infusion of taurocholate into the pancreatic duct. C23, an eCIRP inhibitor, was given 1 h prior to induction of AP. Pancreatic, lung, and blood samples were collected and levels of citrullinated histone 3, DNA-histone complexes, eCIRP, myeloperoxidase (MPO), amylase, cytokines, matrix metalloproteinase-9 (MMP-9), and CXC chemokines were quantified after 24 h. NETs were detected by electron microscopy in the pancreas and bone marrow-derived neutrophils. Amylase secretion was analyzed in isolated acinar cells. Plasma was obtained from healthy individuals and patients with mild and moderate severe or severe AP. Taurocholate infusion induced NET formation, inflammation, and tissue injury in the pancreas. Pretreatment with C23 decreased taurocholate-induced pancreatic and plasma levels of eCIRP and tissue damage in the pancreas. Blocking eCIRP reduced levels of citrullinated histone 3 and NET formation in the pancreas as well as DNA-histone complexes in the plasma. In addition, administration of C23 attenuated MPO levels in the pancreas and lung of mice exposed to taurocholate. Inhibition of eCIRP reduced pancreatic levels of CXC chemokines and plasma levels of IL-6, HMGB-1, and MMP-9 in mice with severe AP. Moreover, eCIRP was found to be bound to NETs. Coincubation with C23 reduced NET-induced amylase secretion in isolated acinar cells. Patients with severe AP had elevated plasma levels of eCIRP compared with controls. Our novel findings suggest that eCIRP is a potent regulator of NET formation in the inflamed pancreas. Moreover, these results show that targeting eCIRP with C23 inhibits inflammation and tissue damage in AP. Thus, eCIRP could serve as an effective target to attenuate pancreatic damage in patients with AP.
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| 8. |
- Purhonen, Janne, et al.
(författare)
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Mitochondrial complex III deficiency drives c-MYC overexpression and illicit cell cycle entry leading to senescence and segmental progeria
- 2023
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Ingår i: Nature Communications. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Accumulating evidence suggests mitochondria as key modulators of normal and premature aging, yet whether primary oxidative phosphorylation (OXPHOS) deficiency can cause progeroid disease remains unclear. Here, we show that mice with severe isolated respiratory complex III (CIII) deficiency display nuclear DNA damage, cell cycle arrest, aberrant mitoses, and cellular senescence in the affected organs such as liver and kidney, and a systemic phenotype resembling juvenile-onset progeroid syndromes. Mechanistically, CIII deficiency triggers presymptomatic cancer-like c-MYC upregulation followed by excessive anabolic metabolism and illicit cell proliferation against lack of energy and biosynthetic precursors. Transgenic alternative oxidase dampens mitochondrial integrated stress response and the c-MYC induction, suppresses the illicit proliferation, and prevents juvenile lethality despite that canonical OXPHOS-linked functions remain uncorrected. Inhibition of c-MYC with the dominant-negative Omomyc protein relieves the DNA damage in CIII-deficient hepatocytes in vivo. Our results connect primary OXPHOS deficiency to genomic instability and progeroid pathogenesis and suggest that targeting c-MYC and aberrant cell proliferation may be therapeutic in mitochondrial diseases.
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| 9. |
- Smolag, Karolina I., et al.
(författare)
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Complement inhibitor factor H expressed by breast cancer cells differentiates CD14+ human monocytes into immunosuppressive macrophages
- 2020
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Ingår i: OncoImmunology. - 2162-4011. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Macrophages are a major immune cell type in the tumor microenvironment, where they display a tumor-supporting phenotype. Factor H (FH) is a complement inhibitor that also plays a role in several cellular functions. To date, the phenotype of monocytes stimulated with FH has been unexplored. We discovered that FH is a survival factor for CD14+ primary human monocytes, promoting their differentiation into macrophages in serum-free medium. This activity was localized to the C-terminal domains of FH and it was inhibited in plasma, indicating that the phenomenon may be most relevant in tissues. FH-induced macrophages display characteristics of immunosuppressive cells including expression of CD163 and CD206, release of the anti-inflammatory cytokine IL-10 and changes in metabolism. Furthermore, FH-induced macrophages express low levels of HLA-DR but high levels of co-inhibitory molecule programmed death-ligand 1 (PD-L1), and accordingly, a reduced capacity for T-cell activation. Finally, we show that FH is expressed by human breast cancer cells and that this correlates with the presence of immunosuppressive macrophages, breast cancer recurrence and severity of the disease. We propose that the expression of FH by tumor cells and the promotion of an immunosuppressive cancer microenvironment by this protein should be taken into account when considering the effectiveness of immunotherapies against breast cancer.
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| 10. |
- Spanou, Chara E.S., et al.
(författare)
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Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation
- 2023
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Ingår i: FASEB Journal. - 0892-6638. ; 37:1
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Tidskriftsartikel (refereegranskat)abstract
- Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.
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