scholarly journals Protocadherin-7 Regulates Osteoclast Differentiation through Intracellular SET-Binding Domain-Mediated RhoA and Rac1 Activation

2021 ◽  
Vol 22 (23) ◽  
pp. 13117
Author(s):  
Hyunsoo Kim ◽  
Noriko Takegahara ◽  
Yongwon Choi
Keyword(s):  
Molecular Mechanisms ◽  
Active Form ◽  
Osteoclast Differentiation ◽  
Small Gtpases ◽  
Binding Domain ◽  
Full Length ◽  
Retroviral Transduction ◽  
Cadherin Superfamily ◽  
Intracellular Region

Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Although the cell-intrinsic role of Pcdh7 in osteoclast differentiation has been demonstrated, the molecular mechanisms of Pcdh7 regulating osteoclast differentiation remain to be determined. Here, we demonstrate that Pcdh7 contributes to osteoclast differentiation by regulating small GTPases, RhoA and Rac1, through its SET oncoprotein binding domain. Pcdh7 is associated with SET along with RhoA and Rac1 during osteoclast differentiation. Pcdh7-deficient (Pcdh7−/−) cells showed abolished RANKL-induced RhoA and Rac1 activation, and impaired osteoclast differentiation. Impaired osteoclast differentiation in Pcdh7−/− cells was restored by retroviral transduction of full-length Pcdh7 but not by a Pcdh7 mutant that lacks SET binding domain. The direct crosslink of the Pcdh7 intracellular region induced the activation of RhoA and Rac1, which was not observed when Pcdh7 lacks the SET binding domain. Additionally, retroviral transduction of the constitutively active form of RhoA and Rac1 completely restored the impaired osteoclast differentiation in Pcdh7−/− cells. Collectively, these results demonstrate that Pcdh7 controls osteoclast differentiation by regulating RhoA and Rac1 activation through the SET binding domain.

The V-ATPase a2-subunit as a putative endosomal pH-sensor

2007 ◽  
Vol 35 (5) ◽  
pp. 1092-1099 ◽  
Author(s):  
V. Marshansky
Keyword(s):  
Crucial Role ◽  
Molecular Mechanisms ◽  
Ph Sensor ◽  
Small Gtpases ◽  
Ph Sensitive ◽  
Ph Sensing ◽  
Degradative Pathway ◽  
Histidine Residues ◽  
Endosomal Acidification

V-ATPase (vesicular H+-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor–ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

Pharmacological Inhibition of Lck is Able to Revert Glucocorticoid Resistance in Pediatric T-ALL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 838-838
Author(s):  
Valentina Serafin ◽  
Giorgia Capuzzo ◽  
Gloria Milani ◽  
Silvia Bresolin ◽  
Marica Pinazza ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Survival ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Resistant Cell ◽  
Poor Responders ◽  
Downstream Target ◽  
Blast Cells ◽  
Approved Drugs

Abstract The AIEOP-BFM group has traditionally used the peripheral blood blast cells count after a 7-day glucocorticoids (GC) prephase to classify patients as Prednisone Good Responders (PGR) or Prednisone Poor Responders (PPR). As described by Schrappe M. in 2011, PPR patients tend to have a worse prognosis, despite the fact that all of them are assigned to the High Risk protocol. Little is known about the molecular mechanisms that lead to GC resistance, guiding our research to the identification of new specific molecular targets in order to develop new approaches to improve therapy efficacy in these patients. To this end, we performed a Reverse Phase Protein Analysis (RPPA) of 54 PGR and 33 PPR pediatric T-ALL patients at diagnosis, and studied the activation or expression of 87 proteins involved in key cellular signaling pathways. Interestingly, we found a higher expression of LCK phosphorylated at Y505 (inhibited form) in PGR patients (p=0.001), together with a higher phosphorylation of SRC Y416 (active form) in PPR patients (p=0.01). Total LCK and LCK RNA expression were not differentially expressed in the two subgroups of patients, suggesting an increased activation of LCK in PPR patients. Indeed, in agreement with these results, also LCK downstream target PLCɣ, phosphorylated at Y783, resulted hyperactivated in PPR compared to PGR patients (p=0.05), confirmed also by a positive correlation between PLCɣ Y783 and SRC Y416 (r=0.51, p=0.01). Taken together, these results indicate a hyperactivation of the LCK pathway in PPR patients compared to PGR ones. LCK is part of the TCR multiprotein complex together with the GC receptor. In normal T lymphocytes, after GC treatment the complex is disrupted, LCK activation is decreased and downstream prosurvival signaling inhibited, thus leading to cell death. In this light, in GC resistant patients hyperactivated LCK might sustain cell survival regardless of GC activity. We then tested if FDA-approved or recently developed LCK inhibitors would revert GC resistance in T-ALL cells. GC resistant cell lines ALL-SIL, T-ALL1 and CEM were treated with Dasatinib, Bosutinib, Nintedanib and WH-4-023 alone or in combination with Dexamethasone (Dex). All four inhibitors alone are able to decrease cell survival, and all of them strongly synergize with Dex, bringing to the sensitization of these cells to GC treatment. We also tested these compounds alone or in combination with Dex in 4 PPR T-ALL patients cells derived from xenograft mice. Also in these cases we observed an enhanced sensitization of cells to GC treatment. Finally, corroborating the crucial role of LCK in GC resistance, we observed a strong decrease in cell viability after specific LCK gene silencing and Dex treatment in ALL-SIL cells, together with an increased GC resistance following LCK hyperactivation in P12-ICHIKAWA GC sensitive cells. Thus, our results strongly suggest that the inhibition of LCK using clinically approved drugs could represent a promising new additional therapeutic strategy to revert drug resistance in high-risk pediatric T-ALL patients. Disclosures Indraccolo: OncoMed Pharmaceuticals, Inc.: Research Funding.

scholarly journals The effect of the lipid-binding site of the ankyrin-binding domain of erythroid β-spectrin on the properties of natural membranes and skeletal structures

2010 ◽  
Vol 15 (3) ◽  
Author(s):  
Anna Chorzalska ◽  
Agnieszka Łach ◽  
Tomasz Borowik ◽  
Marcin Wolny ◽  
Anita Hryniewicz-Jankowska ◽  
...  
Keyword(s):  
Barrier Properties ◽  
Lipid Binding ◽  
Binding Domain ◽  
Full Length ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Ip3 Receptor ◽  
Induced Aggregation

AbstractIt was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na+K+ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.

scholarly journals Multifunctional Role of Choline Binding Protein G in Pneumococcal Pathogenesis

2006 ◽  
Vol 74 (2) ◽  
pp. 821-829 ◽  
Author(s):  
B. Mann ◽  
C. Orihuela ◽  
J. Antikainen ◽  
G. Gao ◽  
J. Sublett ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Natural Variation ◽  
Binding Protein ◽  
Surface Protein ◽  
Binding Domain ◽  
Full Length ◽  
Truncated Protein ◽  
Attached Form

ABSTRACT Members of the choline binding protein (Cbp) family are noncovalently bound to phosphorylcholine residues on the surface of Streptococcus pneumoniae. It has been suggested that CbpG plays a role in adherence and increase virulence both at the mucosal surface and in the bloodstream, but the function of this protein has been unclear. A new sequence analysis indicated that CbpG is a possible member of the S1 family of multifunctional surface-associated serine proteases. Clinical isolates contained two alleles of cbpG, and one-third of the strains expressed a truncated protein lacking the C-terminal, cell wall-anchoring choline binding domain. CbpG on the surface of pneumococci (full length) or released into the supernatant (truncated) showed proteolytic activity for fibronectin and casein, as did CbpG expressed on lactobacilli or as a purified full-length or truncated recombinant protein. Recombinant CbpG (rCbpG)-coated beads adhered to eukaryotic cells, and TIGR4 mutants lacking CbpG or having a truncated CbpG protein showed decreased adherence in vitro and attenuation of disease in mouse challenge models of colonization, pneumonia, and bacteremia. Immunization with rCbpG was protective in an animal model of colonization and sepsis. We propose that CbpG is a multifunctional surface protein that in the cell-attached or secreted form cleaves host extracellular matrix and in the cell-attached form participates in bacterial adherence. This is the first example of distinct functions in virulence that are dependent on natural variation in expression of a choline binding domain.

Impact of the FcγII-receptor on quartz uptake and inflammatory response by alveolar macrophages

2008 ◽  
Vol 294 (6) ◽  
pp. L1137-L1148 ◽  
Author(s):  
Petra Haberzettl ◽  
Roel P. F. Schins ◽  
Doris Höhr ◽  
Verena Wilhelmi ◽  
Paul J. A. Borm ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Alveolar Macrophages ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Inhibitory Effect ◽  
Small Gtpases ◽  
Downstream Signaling ◽  
Particle Uptake

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcγII-receptor (FcγII-R). Fluorescence immunocytochemistry, demonstrating FcγII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcγII-R in DQ12 phagocytosis. FcγII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcγII-R. Consistent with the uptake results, FcγII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-α release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcγII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.

scholarly journals Allosteric activation of preformed EGF receptor dimers by a single ligand binding event

2021 ◽  
Author(s):  
Endang R. Purba ◽  
Ei-ichiro Saita ◽  
Reetesh R. Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Active Form ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane a-helix, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the inactive, symmetric kinase dimer spontaneously activate the receptor in vivo. Optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

scholarly journals Research Progress of the Role of Anthocyanins on Bone Regeneration

2021 ◽  
Vol 12 ◽  
Author(s):  
Wei Mao ◽  
Guowei Huang ◽  
Huan Chen ◽  
Liangliang Xu ◽  
Shengnan Qin ◽  
...  
Keyword(s):  
Bone Regeneration ◽  
Molecular Mechanisms ◽  
Biological Effects ◽  
Continuous Process ◽  
Osteoclast Differentiation ◽  
Fragility Fractures ◽  
Osteoclast Formation ◽  
Research Progress ◽  
Treatment Of Osteoporosis

Bone regeneration in osteoporosis and fragility fractures which are highly associated with age remains a great challenge in the orthopedic field, even though the bone is subjected to a continuous process of remodeling which persists throughout lifelong. Regulation of osteoblast and osteoclast differentiation is recognized as effective therapeutic targets to accelerate bone regeneration in osteopenic conditions. Anthocyanins (ACNs), a class of naturally occurring compounds obtained from colored plants, have received increasing attention recently because of their well-documented biological effects, such as antioxidant, anti-inflammation, and anti-apoptosis in chronic diseases, like osteoporosis. Here, we summarized the detailed research progress on ACNs on bone regeneration and their molecular mechanisms on promoting osteoblast differentiation as well as inhibiting osteoclast formation and differentiation to explore their promising therapeutic application in repressing bone loss and helping fragility fracture healing. Better understanding the role and mechanisms of ACNs on bone regeneration is helpful for the prevention or treatment of osteoporosis and also for the exploration of new bone regenerative medicine.

Crucial polarity regulators in axon specification

2012 ◽  
Vol 53 ◽  
pp. 55-68 ◽  
Author(s):  
Giovanna Lalli
Keyword(s):  
Nervous System ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Membrane Traffic ◽  
Small Gtpases ◽  
Cell Polarization ◽  
Microtubule Cytoskeleton ◽  
Morphological Asymmetry ◽  
Neuronal Polarization

Cell polarization is critical for the correct functioning of many cell types, creating functional and morphological asymmetry in response to intrinsic and extrinsic cues. Neurons are a classical example of polarized cells, as they usually extend one long axon and short branched dendrites. The formation of such distinct cellular compartments (also known as neuronal polarization) ensures the proper development and physiology of the nervous system and is controlled by a complex set of signalling pathways able to integrate multiple polarity cues. Because polarization is at the basis of neuronal development, investigating the mechanisms responsible for this process is fundamental not only to understand how the nervous system develops, but also to devise therapeutic strategies for neuroregeneration. The last two decades have seen remarkable progress in understanding the molecular mechanisms responsible for mammalian neuronal polarization, primarily using cultures of rodent hippocampal neurons. More recent efforts have started to explore the role of such mechanisms in vivo. It has become clear that neuronal polarization relies on signalling networks and feedback mechanisms co-ordinating the actin and microtubule cytoskeleton and membrane traffic. The present chapter will highlight the role of key molecules involved in neuronal polarization, such as regulators of the actin/microtubule cytoskeleton and membrane traffic, polarity complexes and small GTPases.

scholarly journals Elucidating Role of Ezh2 in Tolerogenic Function of NOD Bone Marrow-Derived Dendritic Cells Expressing Constitutively Active Stat5b.

2019 ◽  
Author(s):  
Echarki Zerif ◽  
Denis Gris ◽  
Gilles Dupuis ◽  
Abdelaziz Amrani
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Nod Mice ◽  
Autoreactive T Cell ◽  
Tolerogenic Function ◽  
Constitutively Active

Tolerogenic dendritic cells are crucial to control development of autoreactive T cell responses and prevention of autoimmunity. We have reported that NOD.CD11cStat5b-CA transgenic mice expressing a constitutively active form of Stat5b under the control of CD11c promoter are protected from diabetes and that Stat5b-CA-expressing DCs are tolerogenic and halt ongoing diabetes in NOD mice. However, the molecular mechanisms by which Stat5b-CA modulates DC tolerogenic function is not fully understood. Here, we used bone marrow-derived DCs from NOD.CD11cStat5b-CA transgenic mice (Stat5b-CA.BMDC) and found that Stat5b-CA.BMDC displayed high levels of MHC class II, CD80, CD86, PD-L1 and PD-L2 and produced elevated amounts of TGFβ but low amounts of TNF and IL-23. Stat5b-CA.BMDCs upregulated Irf4 and downregulated Irf8 genes and protein expression and promoted CD11c+CD11b+ DC2 subset differentiation. Interestingly, we found that the histone methyltransferase Ezh2 interacted with Stat5b-CA complex that bound GAS sequences in the Irf8 enhancer whereas Ezh2 did not interact with GAS sequences in the case of the Irf4 promoter. Injection of Stat5b-CA.BMDCs to prediabetic NOD mice halted progression of islet inflammation and protected against diabetes. Importantly, inhibition of Ezh2 in tolerogenic Stat5b-CA.BMDCs reduced their ability to prevent diabetes development in NOD recipient mice. Taken together, our data suggest that the active form of Stat5b induces tolerogenic DC function by modulating IRF4 and IRF8 expression through recruitment of Ezh2 and highlight the fundamental role of Ezh2 in Stat5b-mediated induction of tolerogenic DCs function.

scholarly journals Epigenetic Regulators Involved in Osteoclast Differentiation

2020 ◽  
Vol 21 (19) ◽  
pp. 7080
Author(s):  
Kristina Astleford ◽  
Emily Campbell ◽  
Andrew Norton ◽  
Kim C. Mansky
Keyword(s):  
Elderly Population ◽  
Molecular Mechanisms ◽  
Osteoclast Differentiation ◽  
The Elderly ◽  
Risk Of Fracture ◽  
Multinucleated Cells ◽  
Age Related ◽  
Epigenetic Regulators ◽  
Age Related Changes

Age related changes to the skeleton, such as osteoporosis, increase the risk of fracture and morbidity in the elderly population. In osteoporosis, bone remodeling becomes unbalanced with an increase in bone resorption and a decrease in bone formation. Osteoclasts are large multinucleated cells that secrete acid and proteases to degrade and resorb bone. Understanding the molecular mechanisms that regulate osteoclast differentiation and activity will provide insight as to how hyper-active osteoclasts lead to pathological bone loss, contributing to diseases such as osteoporosis. Reversible modifications to the DNA such as histone acetylation, methylation, phosphorylation and ubiquitylation alters the access of transcriptional machinery to DNA and regulates gene expression and osteoclast differentiation and activity. It is critical for the management of bone related diseases to understand the role of these chromatin modifying proteins during osteoclast differentiation, as potential therapies targeting these proteins are currently under development.

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