scholarly journals Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit

2020 ◽  
Vol 117 (46) ◽  
pp. 28838-28846
Author(s):  
Yunxiao Zhang ◽  
Wan-Jin Lu ◽  
David P. Bulkley ◽  
Jiahao Liang ◽  
Arthur Ralko ◽  
...  
Keyword(s):  
Taste Receptor ◽  
Hedgehog Pathway ◽  
Taste Receptor Cell ◽  
Pathway Activation ◽  
Therapeutic Value ◽  
Tissue Targeting ◽  
Transport Cycle ◽  
Alternative Conformation

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 “switch helix,” as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.

scholarly journals Hedgehog pathway activation through conformational blockade of the Patched sterol conduit

2019 ◽  
Author(s):  
Yunxiao Zhang ◽  
Wan-Jin Lu ◽  
David P. Bulkley ◽  
Jiahao Liang ◽  
Arthur Ralko ◽  
...  
Keyword(s):  
Taste Receptor ◽  
Hedgehog Pathway ◽  
Pathway Activation ◽  
Therapeutic Value ◽  
Tissue Targeting ◽  
Transport Cycle ◽  
Alternative Conformation ◽  
Pharmacologic Agents

AbstractActivation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental and therapeutic application has been difficult for lack of agents amenable to tissue targeting. We have developed a novel agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1. This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 “switch helix”, as revealed by cryo-EM structure determination. Although this conformation likely constitutes part of the transport cycle, nanobody-trapping disrupts the cycle and prevents substrate movement through the Patched1 sterol conduit. Our conformation-selective nanobody approach provides a new route to the development of transporter-related pharmacologic agents and may be generally applicable to the study of other transporters.

scholarly journals Hedgehog pathway activation in T-cell acute lymphoblastic leukemia predicts response to SMO and GLI1 inhibitors

Blood ◽  
2016 ◽  
Vol 128 (23) ◽  
pp. 2642-2654 ◽  
Author(s):  
Antonis Dagklis ◽  
Sofie Demeyer ◽  
Jolien De Bie ◽  
Enrico Radaelli ◽  
Daphnie Pauwels ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Lymphoblastic Leukemia ◽  
Hedgehog Pathway ◽  
Pathway Activation ◽  
Key Points ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Inhibitor Treatment ◽  
Hedgehog Inhibitor

Key Points A subset of T-ALL cases show high expression of hedgehog pathway genes including the SHH ligand and the GLI1 transcription factor. T-ALL samples with high GLI1 expression levels respond to hedgehog inhibitor treatment in vitro and in vivo.

scholarly journals PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Alessandra Giannella ◽  
Giulio Ceolotto ◽  
Claudia Maria Radu ◽  
Arianna Cattelan ◽  
Elisabetta Iori ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Normal Glucose Tolerance ◽  
Calpain Inhibitor ◽  
Pathway Activation ◽  
Normal Glucose ◽  
Circulating Levels ◽  
Dependent Mechanism

Abstract Background Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. Methods In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. Results We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. Conclusions These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

scholarly journals ERK signaling mediates resistance to immunomodulatory drugs in the bone marrow microenvironment

2021 ◽  
Vol 7 (23) ◽  
pp. eabg2697
Author(s):  
Jiye Liu ◽  
Teru Hideshima ◽  
Lijie Xing ◽  
Su Wang ◽  
Wenrong Zhou ◽  
...  
Keyword(s):  
Patient Outcome ◽  
Bone Marrow ◽  
Nuclear Factor Κb ◽  
Mek Inhibitor ◽  
Erk Signaling ◽  
Immunomodulatory Drugs ◽  
Pathway Activation ◽  
Extracellular Signal

Immunomodulatory drugs (IMiDs) have markedly improved patient outcome in multiple myeloma (MM); however, resistance to IMiDs commonly underlies relapse of disease. Here, we identify that tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) knockdown (KD)/knockout (KO) in MM cells mediates IMiD resistance via activation of noncanonical nuclear factor κB (NF-κB) and extracellular signal–regulated kinase (ERK) signaling. Within MM bone marrow (BM) stromal cell supernatants, TNF-α induces proteasomal degradation of TRAF2, noncanonical NF-κB, and downstream ERK signaling in MM cells, whereas interleukin-6 directly triggers ERK activation. RNA sequencing of MM patient samples shows nearly universal ERK pathway activation at relapse on lenalidomide maintenance therapy, confirming its clinical relevance. Combination MEK inhibitor treatment restores IMiD sensitivity of TRAF2 KO cells both in vitro and in vivo. Our studies provide the framework for clinical trials of MEK inhibitors to overcome IMiD resistance in the BM microenvironment and improve patient outcome in MM.

scholarly journals Action of Alpha-Tocopherol Phosphate on Hemolysis in Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
1953 ◽  
Vol 8 (4) ◽  
pp. 336-341 ◽  
Author(s):  
MARJORIE J. WILLIAMS ◽  
MARY P. CLAPP
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Alpha Tocopherol ◽  
Intramuscular Administration ◽  
Coombs Test ◽  
Therapeutic Value

Abstract 1. The action of alpha-tocopherol phosphate (αTPO4), an anticoagulant, on PNH hemolysis was studied by both in vitro and in vivo means. It was found that the addition of αTPO4 to the modified Ham test inhibited lysis of PNH erythrocytes. During the intramuscular administration of αTPO4, the susceptibility of the erythrocytes to lysis by fresh acidified serum progressively decreased. These cells, after incubation with the serum, gave a positive Coombs test. 2. The possible significance of these findings is briefly discussed. 3. It is concluded that αTPO4 has no therapeutic value in PNH.

In vitro and in vivo antioxidant potential of a polyherbal antimalarial as an indicator of its therapeutic value

2014 ◽  
Vol 6 (1) ◽  
pp. 130 ◽  
Author(s):  
Protus Arrey Tarkang ◽  
A.P. Nwakiban Atchan ◽  
J. Kiuate ◽  
F.A. Okalebo ◽  
G.A. Agbor ◽  
...  
Keyword(s):  
Antioxidant Potential ◽  
Therapeutic Value

scholarly journals Hedgehog pathway permissive conditions allow generation of immortal cell lines from granule cells derived from cancerous and non-cancerous cerebellum

Open Biology ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 180145 ◽  
Author(s):  
Constantin Heil
Keyword(s):  
Cell Lines ◽  
Hedgehog Pathway ◽  
Simple Method ◽  
Genetic Ablation ◽  
Neural Lineage ◽  
Shh Pathway ◽  
Pathway Activation

Cerebellar granule cell progenitors (GCPs) undergo proliferation in the post-natal cerebellum that is dependent on sonic hedgehog (SHH) signalling. Deregulated SHH signalling leads to type 2 medulloblastoma (MB). In this work, a novel cell culture protocol is described, which is suitable for the establishment and long-term maintenance of GCP-derived cells. This method is first applied to SHH pathway active MB cells from Atoh1 -cre; Ptch1 FL/FL tumours, which leads to the generation of neurosphere-like cell lines expressing GCP markers and an active SHH signalling pathway. These cells also show high sensitivity to the Smoothened inhibitor vismodegib, therefore recapitulating the SHH pathway requirement for survival shown by type 2 MB. Analysis of culture supplements reveals that bFGF and fetal bovine serum act as inhibitors of the SHH pathway and therefore preclude generation of cell lines that are relevant to the study of the SHH pathway. Consequently, these insights are transferred from the context of MB to non-transformed, post-natal day 7 cerebellum-derived cellular explants. In contrast to other, previously used methods, these GCP cultures proliferate indefinitely and depend on SHH pathway activation, either by means of the small molecule SAG or through genetic ablation of Ptch1 . This culture method therefore leads to the generation of immortal neurosphere-like cell lines, that are named murine SAG-dependent spheres (mSS). Despite long-term culture, mSS cells remain dependent on continuous stimulation of the SHH pathway. Further, mSS cells maintain their lineage after extensive periods in vitro, as demonstrated by their differentiation towards the neural lineage. Herein a simple method for the generation of immortal cell lines from murine cerebella is defined. These lines can be maintained indefinitely through hedgehog pathway activation and maintain the GCP lineage.

scholarly journals Complement alternative pathway activation by chronic lymphocytic leukemia cells: its role in their hepatosplenic localization

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 463-467 ◽  
Author(s):  
F Praz ◽  
G Karsenty ◽  
JL Binet ◽  
P Lesavre
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Alternative Pathway ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Complement Alternative Pathway ◽  
Acetylneuraminic Acid ◽  
Pathway Activation ◽  
Do So

Abstract Using affinity-purified 125I-F(ab')2 anti-human C3, we have investigated the ability of various leukemic cells to activate complement. Lymphocytes from patients with chronic lymphocytic leukemia (CLL) activated the alternative pathway, but cells from patients with other forms of leukemia or normal lymphocytes did not do so. The amount of C3 deposited on the CLL cells was significantly higher in patients with organomegaly (i.e., splenomegaly and/or hepatomegaly). Activation of complement by CLL cells as assessed by C3 deposition on the membrane occurred both in vivo and in vitro and was not related to the N- acetylneuraminic acid content of the membrane.

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