Project description:Biofilms create an impermeable barrier against antimicrobial treatment and immune cell access, severely complicating treatment and clearance of nosocomial Pseudomonas aeruginosa infections. We recently reported that biofilm also contributes to pathogen virulence by regulating the production of the siderophore pyoverdine. In this study, we investigated the role of PqsA, a key cell-signaling protein, in this regulatory pathway. We demonstrate that PqsA promotes pyoverdine production in a biofilm-dependent manner. Under nutritionally deficient conditions, where biofilm and pyoverdine are decoupled, PqsA is dispensable for pyoverdine production. Interestingly, although PqsA-dependent pyoverdine production does not rely upon Pseudomonas quinolone signal (PQS) biosynthesis, exogenous PQS can also trigger biofilm-independent production of pyoverdine. Adding PQS rapidly induced planktonic cell aggregation. Moreover, these clumps of cells exhibit strong expression of pyoverdine biosynthetic genes and show substantial production of this siderophore. Finally, we surveyed the relationship between biofilm formation and pyoverdine production in various clinical and environmental isolates of P. aeruginosa to evaluate the clinical significance of targeting biofilm during infections. Our findings implicate PqsA in P. aeruginosa virulence by regulating biofilm formation and pyoverdine production.
Project description:?-Synuclein (?S) is the primary protein associated with Parkinson's disease, and it undergoes aggregation from its intrinsically disordered monomeric form to a cross-? fibrillar form. The closely related homolog ?-synuclein (?S) is essentially fibril-resistant under cytoplasmic physiological conditions. Toxic gain-of-function by ?S has been linked to dysfunction, but the aggregation behavior of ?S under altered pH is not well-understood. In this work, we compare fibril formation of ?S and ?S at pH 7.3 and mildly acidic pH 5.8, and we demonstrate that pH serves as an on/off switch for ?S fibrillation. Using ?S/?S domain-swapped chimera constructs and single residue substitutions in ?S, we localized the switch to acidic residues in the N-terminal and non-amyloid component domains of ?S. Computational models of ?S fibril structures indicate that key glutamate residues (Glu-31 and Glu-61) in these domains may be sites of pH-sensitive interactions, and variants E31A and E61A show dramatically altered pH sensitivity for fibril formation supporting the importance of these charged side chains in fibril formation of ?S. Our results demonstrate that relatively small changes in pH, which occur frequently in the cytoplasm and in secretory pathways, may induce the formation of ?S fibrils and suggest a complex role for ?S in synuclein cellular homeostasis and Parkinson's disease.
Project description:Contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) play a crucial role in governing calcium regulation and lipid homeostasis. Despite their significance, the factors regulating their spatial distribution on the PM remain elusive. Inspired by observations in cardiomyocytes, where ER-PM contact sites concentrate on tubular PM invaginations known as transverse tubules (T-tubules), we hypothesize that the PM curvature plays a role in ER-PM contact formation. Through precise control of PM invaginations, we show that PM curvatures locally induce the formation of ER-PM contacts in cardiomyocytes. Intriguingly, the junctophilin family of ER-PM tethering proteins, specifically expressed in excitable cells, is the key player in this process, while the ubiquitously expressed extended synaptotagmin 2 does not show a preference for PM curvature. At the mechanistic level, we find that the low complexity region (LCR) and the MORN motifs of junctophilins can independently bind to the PM, but both the LCR and MORN motifs are required for targeting PM curvatures. By examining the junctophilin interactome, we identify a family of curvature-sensing proteins, Eps15-homology domain containing proteins (EHDs), that interact with the MORN_LCR motifs and facilitate junctophilins' preferential tethering to curved PM. These findings highlight the pivotal role of PM curvature in the formation of ER-PM contacts in cardiomyocytes and unveil a novel mechanism for the spatial regulation of ER-PM contacts through PM curvature modulation.
Project description:ObjectivesConnexin-mediated functional gap junction intercellular communication (GJIC) has a vital role in development, homeostasis and pathology. Transforming growth factor-β1 (TGF-β1), as one of the most vital factors in chondrocytes, promotes cartilage precursor cell differentiation and chondrocyte proliferation, migration and metabolism. However, how TGF-β1 mediates GJIC in chondrocytes remains unclear. This study aims to determine the influence of TGF-β1 on GJIC in mouse chondrocytes and its underlying mechanism.MethodsqPCR and mRNA microarray were used to verify the expression of genes in the TGF-β and connexin families in cartilage and chondrocytes. A scrape loading/dye transfer assay was performed to explore GJIC. Western blot analysis was used to detect connexin43 (Cx43) and Smad signalling components. Immunofluorescence staining was performed to characterize protein distribution.ResultsThe TGF-β1 mRNA was the highest expressed member of the TGFβ super family in cartilage. TGF-β1 promoted functional GJIC through increased expression of Cx43. TGF-β1-mediated GJIC required the participation of TGF-β type I receptor. TGF-β1 activated Smad3 and Smad4 signalling to facilitate their nuclear translocation. The Smad3 and Smad4 signalling proteins bound to the promoter of Gja1 and thus initiated Cx43 gene expression.ConclusionsFor the first time, these results revealed a vital role of TGF-β1 in cell-cell communication in chondrocytes via gap junction formation. We describe the regulatory mechanism, the involvement of TGF-β type I receptor and the nuclear translocation of Smad3/4.
Project description:The E3 ubiquitin ligase complex CDC20-activated Anaphase-promoting complex/Cyclosome (APC/CCDC20) plays a critical role in governing mitotic progression by targeting key cell-cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co-activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well-known cell-cycle related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA-binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11-dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell-cycle independent function of CDC20, establishes APC11CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone-related diseases.
Project description:Energy stress, characterized by the reduction of intracellular ATP, has been implicated in various diseases, including cancer. Here, we show that energy stress promotes the formation of P-bodies in a ubiquitin-dependent manner. Upon ATP depletion, the E3 ubiquitin ligase TRIM23 catalyzes lysine-63 (K63)-linked polyubiquitination of HCLS1-associated protein X-1 (HAX1). HAX1 ubiquitination triggers its liquid‒liquid phase separation (LLPS) and contributes to P-bodies assembly induced by energy stress. Ubiquitinated HAX1 also interacts with the essential P-body proteins, DDX6 and LSM14A, promoting their condensation. Moreover, we find that this TRIM23/HAX1 pathway is critical for the inhibition of global protein synthesis under energy stress conditions. Furthermore, high HAX1 ubiquitination, and increased cytoplasmic localization of TRIM23 along with elevated HAX1 levels, promotes colorectal cancer (CRC)-cell proliferation and correlates with poor prognosis in CRC patients. Our data not only elucidate a ubiquitination-dependent LLPS mechanism in RNP granules induced by energy stress but also propose a promising target for CRC therapy.
Project description:AimThis study investigated whether histamine could play a protective role in pathophysiological response of spinal cord injury (SCI) and regulate the glial scar formation.MethodsFunctional assessment and histological analyses were performed to investigate the effect of histamine after SCI. Histidine decarboxylase knockout (HDC(-/-)) mice were used to confirm the action of histamine. Selective antagonists for H1 and H2 receptors were utilized in vivo and in vitro to verify the functional properties of histamine on astrogliosis.ResultsThe local administration of histamine significantly attenuated the tissue damage and glial scar formation after SCI. In particular, the astrogliosis and neurocan expression found around the lesion were significantly suppressed by histamine. Immunofluorescent staining for neurofilament showed that histamine promoted axonal growth across the glial scar. The HDC(-/-) mice, lacking in endogenous histamine, showed lower behavior score, increased lesion size and astrogliosis, as compared with the wild types. The effect of histamine on locomotor recovery and reactive astrogliosis is reversed by H1 receptor antagonist but not H2 receptor antagonist.ConclusionsOur results indicate that histamine significantly improved the chronic locomotor recovery via attenuating astrogliosis after SCI by stimulating histamine H1 receptor. This study highlights a therapeutic potential of histamine and its related drugs for SCI.
Project description:Human induced pluripotent stem cells (hiPSCs) can differentiate into cells of the three germ layers and are promising cell sources for regenerative medicine therapies. However, current protocols generate hiPSCs with low efficiency, and the generated iPSCs have variable differentiation capacity among different clones. Our previous study reported that MYC proteins (c-MYC and MYCL) are essential for reprogramming and germline transmission but that MYCL can generate hiPSC colonies more efficiently than c-MYC. The molecular underpinnings for the different reprogramming efficiencies between c-MYC and MYCL, however, are unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic differences and promote hiPSC generation in MYCL-induced reprogramming. Proteome analyses suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while the MB2 domain regulates RNA processes. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.
Project description:The differences of pork quality characteristics among different pig breeds mainly came from the differences in myofiber type compositions. Growing evidence indicated the key role of miRNAs in myofiber specification. In the present study, we found that miR-152 is more abundant in the slow-twitch myofiber-enriched muscles. However, its role in myofiber type transformation and myogenesis is largely unknown. Overexpression of miR-152 in porcine myotubes promoted the formation of slow-twitch myofibers and myogenesis. While, inhibition of miR-152 expression showed the opposite effect to miR-152 mimics transfection. The luciferase reporter analysis confirmed that miR-152 straightly targets the 3'-untranslated region (3'-UTR) of uncoupling protein 3 (UCP3) to cause its post-transcriptional inhibition in the protein level. The knockdown of UCP3 by siRNA showed the similar effect of miR-152 on myofiber type transition. Furthermore, the rescue experiment in the porcine myotube transfected with miR-152 mimics or/and UCP3 overexpression plasmid with or without the 3'UTR revealed that UCP3 mediates the action of miR-152 in slow-twitch myofiber formation. Taken together, our findings proposed a novel molecular mechanism through which miR-152 epigenetically regulates meat quality via promoting slow-twitch myofiber formation and skeletal myogenesis.
Project description:Instantaneous blood coagulation after bioengineered liver transplantation is a major issue, and the key process in its prevention is the construction of the endothelial vascular bed on biomimetic scaffolds. However, the specific molecules involved in the regulation of the vascular bed formation remain unclear. Syndecan-4 is a type I transmembrane glycoprotein commonly expressed in the human body; its receptor has been reported as critical for optimal cell adhesion and initiation of intracellular signaling, indicating its promising application in vascular bed formation. In the current study, bioinformatics analysis and in vitro experiments were performed to evaluate whether syndecan-4 promoted endothelial cell migration and functional activation. Exogenous syndecan-4-overexpressing endothelial cells were perfused into the decellularized liver scaffold, which was assessed by Masson's trichrome staining. Western blotting and qRT-PCR were used to evaluate the effects of syndecan-4 on the thrombospondin 1 (THBS1) stability. We found that syndecan-4 promoted the adhesion of vascular endothelial cells and facilitated cell migration and angiogenesis. Furthermore, syndecan-4 overexpression resulted in a well-aligned endothelium on the decellularized liver scaffolds. Mechanistically, syndecan-4 destabilized THBS1 at the protein level. Therefore, our data revealed that syndecan-4 promoted the biological activity of endothelial cells on the bionic liver vascular bed through THBS1. These findings provide scientific evidences for solving transient blood coagulation after bionic liver transplantation.