scholarly journals PHARMACOLOGICAL PROTEIN INACTIVATION BY TARGETING FOLDING INTERMEDIATES

2020 ◽  
Author(s):  
Giovanni Spagnolli ◽  
Tania Massignan ◽  
Andrea Astolfi ◽  
Silvia Biggi ◽  
Paolo Brunelli ◽  
...  
Keyword(s):  
Protein Expression ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Surface Glycoprotein ◽  
Folding Pathway ◽  
Biological Regulation ◽  
Cell Surface Glycoprotein ◽  
Folding Intermediates ◽  
Protein Inactivation ◽  
Small Molecule Ligands

ABSTRACTRecent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

scholarly journals Pharmacological inactivation of the prion protein by targeting a folding intermediate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Giovanni Spagnolli ◽  
Tania Massignan ◽  
Andrea Astolfi ◽  
Silvia Biggi ◽  
Marta Rigoli ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Surface Glycoprotein ◽  
Folding Intermediate ◽  
Biological Regulation ◽  
Cell Surface Glycoprotein ◽  
Folding Intermediates ◽  
Protein Levels ◽  
Small Molecule Ligands

AbstractRecent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

scholarly journals A Strong Expression of CD44-6v Correlates With Shorter Survival of Patients With Acute Myeloid Leukemia

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3401-3413 ◽  
Author(s):  
S. Legras ◽  
U. Günthert ◽  
R. Stauder ◽  
F. Curt ◽  
S. Oliferenko ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Expression ◽  
Myeloid Leukemia ◽  
Surface Glycoprotein ◽  
Leukemic Cells ◽  
Cell Surface Glycoprotein ◽  
Variant Isoforms ◽  
Cd34 Cells ◽  
First Time ◽  
Acute Myeloid

CD44 is a ubiquitous cell-surface glycoprotein that displays many variant isoforms (CD44v) generated by alternative splicing of exons 2v to 10v. The expression of variant isoforms is highly restricted and correlated with specific processes, such as leukocyte activation and malignant transformation. We have herein studied CD44v expression in acute myeloid leukemia (AML) and, for comparison, in normal myelopoiesis. Protein expression of total CD44 and of CD44-3v, -6v, and -9v isoforms has been measured using specific monoclonal antibodies and flow cytometry. The composition of variant exon transcripts has been analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction followed by Southern hybridization with exon-specific probes. Our data show that (1) CD44-6v isoforms are expressed on 12.0% ± 2.5% of normal CD34+ cells; this expression is sharply upregulated through monopoiesis and, inversely, downregulated during granulopoiesis. Also, CD44-3v and CD44-9v isoforms are detected on 10% and 14% of normal monocytes, respectively. (2) Sixty-nine from a total of 95 AML patients display a variable proportion (range, 5% to 80%) of CD44-6v+ leukemic cells. (3) A shorter overall survival characterizes the group of AML patients displaying more than 20% of CD44-6v+ leukemic cells (8 months v 18 months, P < .02). These data suggest, for the first time, that the protein expression of CD44-6v containing isoforms may serve as a new prognostic factor in AML.

scholarly journals Limited Value of KAI1/CD82 Protein Expression as a Prognostic Marker in Human Gastric Cancer

2012 ◽  
Vol 32 (6) ◽  
pp. 337-342 ◽  
Author(s):  
Maximilian Knoener ◽  
Till Krech ◽  
Florian Puls ◽  
Ulrich Lehmann ◽  
Hans Kreipe ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Protein Expression ◽  
Tumor Growth ◽  
Prognostic Marker ◽  
Metastatic Lymph Node ◽  
Surface Glycoprotein ◽  
Human Gastric Cancer ◽  
Cell Surface Glycoprotein ◽  
Gastric Carcinomas ◽  
Tumor Growth And Metastasis

The cell surface glycoprotein KAI1/CD82 suppresses tumor growth and metastasis in animal models. This study aimed to evaluate the prognostic relevance of KAI1/CD82 protein expression in human gastric cancer. Primary gastric carcinomas (n= 271) with amean clinical follow-up time of 48months were immunostained using the monoclonal anti-KAI1/CD82 antibody G2. Staining was evaluated as negativeversuspositive for statistical analysis. KAI1/CD82 immunoreactivity was absent in 103/271 (38%) cases. There was a trend towards KAI1/CD82 negativity in poorly differentiated cases (p= 0.0679). Moreover, KAI1/CD82-negative carcinomas were associated with a higher pT status (p= 0.0222), metastatic lymph node involvement (p= 0.0018) and a higher clinical tumor stage (p= 0.0050). The median overall survival times of KAI1/CD82-negative and KAI1/CD82-positive gastric carcinomas were 20 and 37 months, respectively (p= 0.2305). These results are in line with the proposed function of KAI1/CD82 as a suppressor of tumor growth and metastasis. However, these data suggest that KAI1/CD82, as detected by immunohistochemistry, is of limited value as a prognostic marker for gastric cancer in routine histological workup.

scholarly journals Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor

Retrovirology ◽  
2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Kryštof Štafl ◽  
Martin Trávníček ◽  
Dana Kučerová ◽  
Ľubomíra Pecnová ◽  
Veronika Krchlíková ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Large Scale ◽  
Complex Structure ◽  
Surface Glycoprotein ◽  
Soluble Form ◽  
Cell Surface Glycoprotein ◽  
Enveloped Virus ◽  
Set Up ◽  
Cell Cell

Abstract Background Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell–cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. Results We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell–cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. Conclusions We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.

scholarly journals CD26/DPPIV Signal Transduction Function, but Not Proteolytic Activity, Is Directly Related to Its Expression Level on Human Th1 and Th2 Cell Lines as Detected with Living Cell Cytochemistry

2002 ◽  
Vol 50 (9) ◽  
pp. 1169-1177 ◽  
Author(s):  
Emil P. Boonacker ◽  
Eddy A. Wierenga ◽  
Hermelijn H. Smits ◽  
Cornelis J.F. Van Noorden
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Glycoprotein ◽  
Th2 Cells ◽  
Cresyl Violet ◽  
T Helper 1 ◽  
Cell Surface Glycoprotein ◽  
A Cell ◽  
Th1 And Th2

CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was specifically determined with the synthetic fluorogenic substrate ala-pro-cresyl violet and CD26 protein expression was demonstrated with an FITC-conjugated CD26-specific antibody. Fluorescence of liberated cresyl violet (red) and FITC (green) was detected simultaneously on living T-cells using flow cytometry and spectrofluorometry. Th1 cells expressed three- to sixfold more CD26 protein than Th2 cells. The signal transduction function of the CD26/DPPIV complex, tested by measuring its co-stimulatory potential for proliferation, was directly related to the amount of CD26 protein at the cell surface. However, DPPIV activity was similar in both cell populations at physiological substrate concentrations because of differences in Km and Vmax values of DPPIV on Th1 and Th2 cells. Western blotting and zymography of Th1 and Th2 whole-cell lysates demonstrated similar patterns. This study shows that two functions of one molecule can be controlled differentially.

scholarly journals The Cellular Prion Protein: A Promising Therapeutic Target for Cancer

2020 ◽  
Vol 21 (23) ◽  
pp. 9208
Author(s):  
Gyeongyun Go ◽  
Sang Hun Lee
Keyword(s):  
Drug Resistance ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Tumor Progression ◽  
Prion Protein ◽  
Immune Modulation ◽  
Cellular Prion Protein ◽  
Surface Glycoprotein ◽  
Stem Cell Marker ◽  
Cell Surface Glycoprotein

Studies on the cellular prion protein (PrPC) have been actively conducted because misfolded PrPC is known to cause transmissible spongiform encephalopathies or prion disease. PrPC is a glycophosphatidylinositol-anchored cell surface glycoprotein that has been reported to affect several cellular functions such as stress protection, cellular differentiation, mitochondrial homeostasis, circadian rhythm, myelin homeostasis, and immune modulation. Recently, it has also been reported that PrPC mediates tumor progression by enhancing the proliferation, metastasis, and drug resistance of cancer cells. In addition, PrPC regulates cancer stem cell properties by interacting with cancer stem cell marker proteins. In this review, we summarize how PrPC promotes tumor progression in terms of proliferation, metastasis, drug resistance, and cancer stem cell properties. In addition, we discuss strategies to treat tumors by modulating the function and expression of PrPC via the regulation of HSPA1L/HIF-1α expression and using an anti-prion antibody.

scholarly journals A Strong Expression of CD44-6v Correlates With Shorter Survival of Patients With Acute Myeloid Leukemia

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3401-3413 ◽  
Author(s):  
S. Legras ◽  
U. Günthert ◽  
R. Stauder ◽  
F. Curt ◽  
S. Oliferenko ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Expression ◽  
Myeloid Leukemia ◽  
Surface Glycoprotein ◽  
Leukemic Cells ◽  
Cell Surface Glycoprotein ◽  
Variant Isoforms ◽  
Cd34 Cells ◽  
First Time ◽  
Acute Myeloid

Abstract CD44 is a ubiquitous cell-surface glycoprotein that displays many variant isoforms (CD44v) generated by alternative splicing of exons 2v to 10v. The expression of variant isoforms is highly restricted and correlated with specific processes, such as leukocyte activation and malignant transformation. We have herein studied CD44v expression in acute myeloid leukemia (AML) and, for comparison, in normal myelopoiesis. Protein expression of total CD44 and of CD44-3v, -6v, and -9v isoforms has been measured using specific monoclonal antibodies and flow cytometry. The composition of variant exon transcripts has been analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction followed by Southern hybridization with exon-specific probes. Our data show that (1) CD44-6v isoforms are expressed on 12.0% ± 2.5% of normal CD34+ cells; this expression is sharply upregulated through monopoiesis and, inversely, downregulated during granulopoiesis. Also, CD44-3v and CD44-9v isoforms are detected on 10% and 14% of normal monocytes, respectively. (2) Sixty-nine from a total of 95 AML patients display a variable proportion (range, 5% to 80%) of CD44-6v+ leukemic cells. (3) A shorter overall survival characterizes the group of AML patients displaying more than 20% of CD44-6v+ leukemic cells (8 months v 18 months, P < .02). These data suggest, for the first time, that the protein expression of CD44-6v containing isoforms may serve as a new prognostic factor in AML.

Rapid Quantification of Prion Proteins

2019 ◽  
Author(s):  
Matthew Healey ◽  
Muttuswamy Sivakumaran ◽  
Mark Platt
Keyword(s):  
Prion Proteins ◽  
Prion Diseases ◽  
Sample Volume ◽  
Cellular Prion Protein ◽  
Dna Aptamer ◽  
Superparamagnetic Beads ◽  
Resistive Pulse ◽  
Large Sample Volume ◽  
Complex Sample ◽  
Neurological Conditions

<p>Prion diseases are a group of fatal transmissible neurological conditions caused by the change in conformation of the normal intrinsic cellular prion protein (PrP<sup>C</sup>) in to the highly ordered insoluble amyloid state conformer (PrP<sup>SC</sup>). We present a rapid assay using Aptamers and Resistive Pulse Sensing, RPS, to extract and quantify proteins from complex sample matrices, demonstrate with the quantification of PrP<sup>c</sup>. We functionalise the surface of superparamagnetic beads, SPBs, with a DNA aptamer. First SPB’s termed P-Beads, are used to pre-concentrate the analyte from a large sample volume. The PrP<sup>c</sup> protein is then eluted from the P-Beads before aptamer modified sensing beads, S-Beads, are added. The velocity of the S-Beads through the nanopore reveals the concentration of the PrP<sup>c</sup> protein. The process is done in under an hour and allows the detection of picomol’s of protein. The technique could be easily adopted to the mutated version of the protein and integrated into clinical workflows for the screening of blood donations and transfusions. </p>

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