Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro

Besides its insulinotropic actions on pancreatic β cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron–IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.

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JAK1 palmitoylation by ZDHHC3/7 is Essential for Neuropoietic Cytokine Signaling and DRG Neuron Survival

10.1101/2020.11.16.385971 ◽

2020 ◽

Author(s):

Luiselys M. Hernandez ◽

Audrey Montersino ◽

Jingwen Niu ◽

Shuchi Guo ◽

Gareth M. Thomas

Keyword(s):

Kinase Activity ◽

Neuronal Development ◽

Janus Kinase ◽

Cytokine Signaling ◽

Drg Neurons ◽

Drg Neuron ◽

Janus Kinase 1 ◽

Survival Signaling ◽

Key Sites

AbstractJanus Kinase-1 (JAK1) plays key roles in pro-survival signaling during neurodevelopment and in responses to neuronal injury. JAK1 was identified as a potential palmitoyl-protein in high-throughput studies, but the importance of palmitoylation for JAK1’s roles in neurons has not been addressed. Here, we report that JAK1 is endogenously palmitoylated in cultured Dorsal Root Ganglion (DRG) neurons and, using an shRNA knockdown/rescue approach, reveal that JAK1 palmitoylation is important for neuropoietic cytokine-dependent signaling and neuronal survival. We further identify the related palmitoyl acyltransferases (PATs) ZDHHC3 and ZDHHC7 as dominant regulators of JAK1 palmitoylation in transfected non-neuronal cells and endogenously in neurons. At the molecular level, palmitoylation is critical for JAK1’s kinase activity in transfected cells and even in vitro, likely because palmitoylation facilitates transphosphorylation of key sites in the JAK1 activation loop. These findings provide new insights into palmitoylation-dependent control of neuronal development and survival.

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Immunoenzymatic demonstration of the simultaneous presence of transferrin, hemopexin and albumin in a same hepatocyte and their ultrastructural localization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081309 ◽

1978 ◽

Vol 36 (2) ◽

pp. 88-89

Author(s):

M. Kraemer ◽

J. Foucrier ◽

J. Vassy ◽

M.T. Chalumeau

Keyword(s):

Plasma Proteins ◽

Rat Hepatocytes ◽

Ultrastructural Localization ◽

Carrier Proteins ◽

Normal Cells ◽

Adult Rat ◽

Adult Rat Hepatocytes ◽

Monospecific Antisera ◽

Different Levels

Some authors using immunofluorescent techniques had already suggested that some hepatocytes are able to synthetize several plasma proteins. In vitro studies on normal cells or on cells issued of murine hepatomas raise the same conclusion. These works could be indications of an hepatocyte functionnal non-specialization, meanwhile the authors never give direct topographic proofs suitable with this hypothesis.The use of immunoenzymatic techniques after obtention of monospecific antisera had seemed to us useful to bring forward a better knowledge of this problem. We have studied three carrier proteins (transferrin = Tf, hemopexin = Hx, albumin = Alb) operating at different levels in iron metabolism by demonstrating and localizing the adult rat hepatocytes involved in their synthesis.Immunological, histological and ultrastructural methods have been described in a previous work.

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Specific Labeling of Protein Domains with Antibody Fragments

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098587 ◽

1981 ◽

Vol 39 ◽

pp. 416-417

Author(s):

U. Aebi ◽

L.E. Buhle ◽

W.E. Fowler

Keyword(s):

Image Processing ◽

Specific Protein ◽

Antibody Fragments ◽

Supramolecular Organization ◽

Two Dimensional ◽

Ordered Structures ◽

Virus Capsids ◽

Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

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Observations on the in vitro Effects of Chylomicra, Low-Density Lipoproteins and Phospholipids on Human Plasma Euglobulin Lysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654971 ◽

1963 ◽

Vol 09 (01) ◽

pp. 164-174 ◽

Cited By ~ 2

Author(s):

Albert R Pappenhagen ◽

J. L Koppel ◽

John H Olwin

Keyword(s):

Human Plasma ◽

Phosphatidyl Ethanolamine ◽

Phosphatidyl Inositol ◽

Plasma Lipoproteins ◽

Low Density ◽

Inhibitory Effects ◽

Soybean Phospholipids ◽

In Vitro Effects ◽

Complete Precipitation

SummaryData have been presented on the in vitro effects of human chylomicra, low-density human plasma lipoproteins, and partially purified preparations of various phospholipids on human plasma euglobulin lysis. Euglobulin lysis was found to be accelerated by preparations of mixed soybean phospholipids (aso-lectin), cephalin, phosphatidyl inositol, phophatidyl serine and phosphatidyl ethanolamine. In contrast, it was found to be inhibited by preparations of human chylomicra, low-density human plasma liproproteins and lecithin. Inhibition of euglobulin lysis produced by any of these three agents could be diminished or completely overcome by the simultaneous presence of suitable levels of any one of the accelerating agents. In all cases studied, both inhibitory and accelerating effects were observed to be concentration-dependent. Evidence has been obtained to suggest that in the case of the accelerating agents the observed increased rate of euglobulin lysis is not a direct effect on lysis itself, but rather is due to more complete precipitation of plasminogen in the presence of these substances. On the other hand, it appears that the inhibitory effects observed are not related to the extent of plasminogen precipitation, but are actually true inhibitions of euglobulin lysis. The possible clinical significance of some of these observations has been briefly discussed.

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EVALUATION OF TISSUE STEROID BINDING IN VITRO

Acta Endocrinologica ◽

10.1530/acta.0.068s223 ◽

1971 ◽

Vol 68 (1_Suppl) ◽

pp. S223-S246 ◽

Cited By ~ 2

Author(s):

C. R. Wira ◽

H. Rochefort ◽

E. E. Baulieu

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Experimental System ◽

Specific Protein ◽

Coupling Mechanism ◽

Target Tissue ◽

Protein Binding Sites ◽

Definition Of ◽

Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

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Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)66942-x ◽

1986 ◽

Vol 261 (31) ◽

pp. 14797-14803 ◽

Cited By ~ 5

Author(s):

T Akiyama ◽

T Kadowaki ◽

E Nishida ◽

T Kadooka ◽

H Ogawara ◽

...

Keyword(s):

Protein Kinases ◽

Specific Protein ◽

Cytoskeletal Proteins ◽

Substrate Specificities

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DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.254 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii63-ii63

Author(s):

Lakshmi Bollu ◽

Derek Wainwright ◽

Lijie Zhai ◽

Erik Ladomersky ◽

Kristen Lauing ◽

...

Keyword(s):

Protein Degradation ◽

Time Course ◽

Immune Cell ◽

Enzyme Inhibitors ◽

Tumor Development ◽

Ubiquitin Proteasome System ◽

Degradation Pathway ◽

Specific Protein ◽

Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

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Interaction of Plasmodium falciparum apicortin with α- and β-tubulin is critical for parasite growth and survival

Scientific Reports ◽

10.1038/s41598-021-83513-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Malabika Chakrabarti ◽

Nishant Joshi ◽

Geeta Kumari ◽

Preeti Singh ◽

Rumaisha Shoaib ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Specific Protein ◽

Parasite Growth ◽

Growth And Survival ◽

Apicomplexan Parasites ◽

Parasite Replication ◽

Main Components ◽

Β Tubulin

AbstractCytoskeletal structures of Apicomplexan parasites are important for parasite replication, motility, invasion to the host cell and survival. Apicortin, an Apicomplexan specific protein appears to be a crucial factor in maintaining stability of the parasite cytoskeletal assemblies. However, the function of apicortin, in terms of interaction with microtubules still remains elusive. Herein, we have attempted to elucidate the function of Plasmodium falciparum apicortin by monitoring its interaction with two main components of parasite microtubular structure, α-tubulin-I and β-tubulin through in silico and in vitro studies. Further, a p25 domain binding generic drug Tamoxifen (TMX), was used to disrupt PfApicortin-tubulin interactions which led to the inhibition in growth and progression of blood stage life cycle of P. falciparum.

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Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature

Scientific Reports ◽

10.1038/s41598-021-81040-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Graziana Esposito ◽

Bijorn Omar Balzamino ◽

Egidio Stigliano ◽

Filippo Biamonte ◽

Andrea Urbani ◽

...

Keyword(s):

Nuclear Translocation ◽

Cell Signalling ◽

Specific Protein ◽

Conditioned Media ◽

Cell Extracts ◽

Dose Dependent Decrease ◽

Significant Expression ◽

Biomolecular Analysis ◽

Protein Signature

AbstractWe previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines’ signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1–100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors’ activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.

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