08/22/2024
By Kwok Fan Chow

The Kennedy College of Science, Department of Chemistry, invites you to attend a Ph.D. Research Proposal defense by Yenping Chang entitled “Physicochemical Characterization of a Recombinant Apolipoprotein and its Lipidated Form.”

Degree: Doctoral
Location: Perry Hall, Room 415
Date: Friday, Aug. 30, 2024
Time: 10:30 a.m.

Committee:

  • Chair Jin Xu, Department of Chemistry, University of Massachusetts Lowell
  • Matthew Gage, Department of Chemistry, University of Massachusetts Lowell
  • Carl Lawton, Department of Chemical Engineering, University of Massachusetts Lowell
  • Dongming Xie, Department of Chemical Engineering, University of Massachusetts Lowell

Abstract:
Apolipoproteins are pivotal for lipid metabolism and cardiovascular health, and their roles extend to diverse functions in the central nervous system (CNS), with implications for neurodegenerative diseases such as Alzheimer's disease (AD). Apolipoprotein E (apoE), a crucial glycoprotein in CNS lipid metabolism, particularly its apoE4 isoform, significantly influences AD pathogenesis by affecting lipid-binding properties and impairing Aβ clearance mechanisms. Structural analysis of apoE reveals domains critical for interactions with lipids and receptors, particularly in AD where apoE4 exacerbates amyloid-beta (Aβ) aggregation and disrupts synaptic function. These insights underscore apoE as a promising target for therapeutic interventions in CNS disorders, and the need to characterize its unique structural attributes.

To establish a comprehensive characterization strategy for apolipoprotein and lipoprotein-like bio-products, this research will employ high performance liquid chromatography (HPLC) to separate the structural isoforms of recombinant ApoE3 based on both hydrophobicity and hydrodynamic size. Initially, hydrophobicity-based methods will be utilized for structural analysis of purified recombinant apoE, facilitating the development of Liquid Chromatography-Mass Spectrometry (LC-MS) methods. Mass spectrometry will enable characterization at both intact and peptide map levels, particularly for revealing and quantifying posttranslational modifications (PTMs) and degradations. Additionally, size exclusion chromatography (SEC) will be employed to characterize protein size distribution under both native and denaturing conditions. Under native conditions, a nanodisc assembly, comprising a lipid bilayer encircled by membrane scaffold proteins (MSP), preserves protein function and offers a natural lipid environment facilitating native analysis of membrane proteins. However, analyzing lipidated complexes is challenging due to the limitations of conventional detection methods, such as UV-Vis spectroscopy, which is not effective for quantifying lipids or complex macromolecules directly. To overcome these limitations, the 3-Detector (3-D) method will be employed, which integrates UV-Vis detection with multi-angle light scattering (MALS), measuring the intensity of scattered light at multiple angles, and differential refractive index (dRI) detector, measuring the difference in refractive index between solution and samples. By leveraging this advanced light scattering technique, the 3-D method provides insights into the lipidation and oligomerization states of apoE in nanodisc assembly. The study also aims to explore SEC under denaturing conditions to investigate the aggregation behavior of apoE, despite challenges with denaturing buffers and HPLC instrument compatibility. Overall, this study will provide comprehensive insights into the structure of apoE proteins and lipidated apoE, guiding the process and formulation development of recombinant ApoE products.

All interested students and faculty members are invited to attend.