Archana Mishra
Archana Mishra
Project: WNT MEMBRANE - Function-oriented plasma membrane proteomic profiling of Chronic Lymphocytic Leukaemia (CLL)
Person in Charge: Mgr. Vítězslav Bryja, Ph.D.
Host institution: Institute of Experimental Biology, Faculty of Science, Masaryk University
Country of Origin: India
Country of scientific activity: Taiwan
Project duration: 24 months
Scientific panel: Life sciences
Abstract:
In this proposal we aim to identify the plasma membrane (PM) and associated proteins involved specifically in B-cells of patients with chronic lymphocytic leukemia (CLL) and not in the healthy B-cells. The alterations occurring at the PM at the CLL cells will be further explored on molecular and functional level. To gain insights into comprehensive membrane protein changes during CLL, we plan to perform a comparative proteomic analysis using two proteomic methods – quantitative proteomics of plasma membrane and immunoprecipitation coupled to mass-spectrometry (IP/MS) of selected membrane proteins related to the WNT pathway (Ror1, Celsr1, Vangl2 and Frizzleds). Plasma membranes of B-cells from healthy/non-progressive/progressive patients, or from cells stimulated with extracellular stimuli (with Wnt, chemokine or both) will be analyzed. We presume that by using proteomic approach we will be able to identify some receptor and effector molecule bridging Wnt signaling pathway (canonical and Non-cononical) with CLL disease progression. We believe that elucidating the profile of extracellular integral membrane proteins on live cells will be vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates.
The ongoing project summary to be published
Our ongoing project is focused on studying molecular mechanisms of non-canonical Wnt signaling in human disease, B-cell chronic lymphocyte leukaemia (CLL). We are using membrane proteomics and immunoprecipitation coupled to Mass-spectrometry (IP/MS) as our main tool to identify proteins (genes) from diseased plasma membrane (PM) involved specifically in B-cells of patients suffering from CLL. We aim to identify and establish disease induced proteomic alterations in the form of comprehensive membrane protein changes occurring at the PM of the CLL cells and further explore the molecular and functional levels at non- canonical Wnt Pathway called planar cell polarity (Wnt/PCP) to establish the identified candidates as a novel CLL biomarker proteins. We use plasma membranes of B-cells from healthy/non-progressive/ progressive CLL patients as the study material.
Summary of results:
In the mentioned period of time we worked to establish an efficient plasma membrane extraction protocol using MEC-1 cells. We used Pierce cell surface protein isolation kit, No. 89881 (Thermo scientific) and implemented membrane proteomics methods and protocol by Peirce and Wait 2009 as a reference to enrich the membrane proteins. The samples obtained using this and IP/MS methods were identified using qTOF mass spectrometry and the identified group of proteins resulted into trace amount of (15-20%) of membrane and membrane associated proteins in total protein sample along with nuclear, ribosomal and cytosolic proteins as major contaminants group. We are currently trying to improve our skills by applying relevant changes at membrane protein isolation steps. We are also trying different solubilization buffers which might further help obtain membrane proteins as major protein group from q-TOF identified proteins.
Secondly, as a key objective of the proposed project we continued to identify composition of membrane protein complexes composed from PCP protein in at CLL condition. Our data showed that several membrane components of Wnt/PCP pathway are strongly up regulated in CLL (reference). As a continuation we performed IP/MS based scans of samples from healthy and melignant CLL induced B-cells. Till date we successfully used Ror1-AF2000 (R&D systems) and DVL3 (santacruz, sc-8027) antibody as baits and immunoprecipitated endogenous binders from five CLL patients B-cell proteins. After successfully detection from LC-MS/MS Q-TOF (Waters) we identified several hits as protein candidates appearing repeatedly in CLL patients. For further results and validation see Part 1.2.
Potential impact and use of Project:
From our till date study and proteomic results results we propose two important directions to study and through light into progression of this chronic disease CLL. 1st-Clinical approach to analyze and propose new gene/protein biomarker candidates of CLL involved in the WNT/PCP signaling. 2nd- In depth functional and molecular analysis of the scrutinized candidates to show and establish their involvement in Wnt/PCP regulated pathway leading to CLL in humans, its progression and cure.
We prove to be successfully using advanced proteomic techniques: Quantitative proteomics based on in solution digestion, which is applied to study CLL for the first time. We believe that elucidating the profile of mass hit candidates will be vital for uncovering and establish them as a diagnostic CLL disease biomarkers, therapeutic agents and drug receptor candidates. We hope this research work might contribute to understand and improve the therapeutic approach following regulating the progression off this chronic disease CLL.