ABSTRACT
Cobll1 affects blast crisis (BC) progression and tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML). PACSIN2, a novel Cobll1 binding protein, activates TKI-induced apoptosis in K562 cells, and this activation is suppressed by Cobll1 through the interaction between PACSIN2 and Cobll1. PACSIN2 also binds and inhibits SH3BP1 which activates the downstream Rac1 pathway and induces TKI resistance. PACSIN2 competitively interacts with Cobll1 or SH3BP1 with a higher affinity for Cobll1. Cobll1 preferentially binds to PACSIN2, releasing SH3BP1 to promote the SH3BP1/Rac1 pathway and suppress TKI-mediated apoptosis and eventually leading to TKI resistance. Similar interactions among Cobll1, PACSIN2, and SH3BP1 control hematopoiesis during vertebrate embryogenesis. Clinical analysis showed that most patients with CML have Cobll1 and SH3BP1 expression at the BC phase and BC patients with Cobll1 and SH3BP1 expression showed severe progression with a higher blast percentage than those without any Cobll1, PACSIN2, or SH3BP1 expression. Our study details the molecular mechanism of the Cobll1/PACSIN2/SH3BP1 pathway in regulating drug resistance and BC progression in CML.
Subject(s)
Adaptor Proteins, Signal Transducing , GTPase-Activating Proteins , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Transcription Factors , Humans , Adaptor Proteins, Signal Transducing/genetics , Apoptosis , Blast Crisis , Drug Resistance , Drug Resistance, Neoplasm , GTPase-Activating Proteins/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Protein Kinase Inhibitors/pharmacology , Transcription Factors/geneticsABSTRACT
There are three subtypes of vertebrate inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), a Ca2+-release channel on the ER membrane - IP3R1, IP3R2, and IP3R3 - each of which has a distinctive role in disease development. To determine the subtype-specific IP3-binding mechanism, we compared the thermodynamics, thermal stability, and conformational dynamics between the N-terminal regions of IP3R1 (IP3R1-NT) and IP3R3 (IP3R3-NT) by performing circular dichroism (CD), isothermal titration calorimetry (ITC), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Previously determined crystal structures of IP3R1-NT and HDX-MS results from this study revealed that both IP3R1 and IP3R3 adopt a similar IP3-binding mechanism. However, several regions, including the α- and ß-interfaces, of IP3R1-NT and IP3R3-NT show significantly different conformational dynamics upon IP3 binding, which may explain the different IP3-binding affinities between the subtypes. The importance of the interfaces for subtype-specific IP3 binding is also supported by the different dynamic conformations of the two subtypes in the apo-states. Furthermore, IP3R1-NT and IP3R3-NT show different IP3-binding affinities and thermal stabilities, but share similar thermodynamic properties for IP3 binding. These results collectively provide new insights into the mechanism underlying IP3 binding to IP3Rs and the subtype-specific regulatory mechanism.
Subject(s)
Inositol 1,4,5-Trisphosphate Receptors/metabolism , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Cell Line , Endoplasmic Reticulum/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors/genetics , Mass Spectrometry , Mice , Models, Biological , Signal Transduction/genetics , Signal Transduction/physiology , ThermodynamicsABSTRACT
Shigella flexneri is a Gram-negative, anaerobic bacterium in the genus Shigella that can cause diarrhoea in humans. SF173, a hypothetical protein from S. flexneri 5a strain M90T, has been cloned, overexpressed, purified and crystallized as a part of laboratory-scale structural genomics project. The SF173 protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.8â M succinic acid pH 7.0 at 293â K. Preliminary X-ray diffraction analysis revealed that the crystal diffracted to 1.47â Å resolution and belonged to space group I432, with unit-cell parameters a=b=c=110.245â Å.
