Analysis, fractionation and exploratory recombinant expression of novel eukaryotic l/D peptide isomerases and free D-amino acid racemases

Andersen IV, Harvey Marvin

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https://hdl.handle.net/2142/120283 Copy

Description

Title

Analysis, fractionation and exploratory recombinant expression of novel eukaryotic l/D peptide isomerases and free D-amino acid racemases

Author(s)

Andersen IV, Harvey Marvin

Issue Date

2023-04-20

Director of Research (if dissertation) or Advisor (if thesis)

Sweedler, Jonathan V

Doctoral Committee Chair(s)

Sweedler, Jonathan V

Committee Member(s)

• Brieher, William M
• Raetzman, Lori T.
• Gillette, Rhanor

Department of Study

Molecular & Integrative Physl

Discipline

Molecular & Integrative Physi

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Peptide isomerase, D-amino acid racemase, LCMS

Abstract

The study of bioactive peptides plays a critical role in advancing our understanding of physiology and the development of medicine. These peptides modulate organism homeostasis, behavior, learning and memory. Over the past 50 years, a vast number of peptides have been characterized through an analysis of primary structure and post-translational modifications. Such an analysis is essential toward elucidating the function of these peptides, as post-translational/co-translational modifications endow these peptides with flexibilities in target recognition and metabolic/chemical stability. As these modifications are largely carried about by peptide processing enzymes, studies to characterize and identify such enzymes are crucial to an investigation of the physiological actions and prevalence of their post-translationally modified peptide substrates. A subtle post-translational modification is the enzymatic conversion of a ribosomally generated peptide into a D-amino acid containing peptide (DAACP). Compared to their all L-amino acid epimers, DAACPs can exhibit differential chemical stability and receptor binding characteristics. They have also been shown to be prevalent across phylogenetically diverse species and occur in a wide array of functional contexts, from venom peptides to neuropeptides. To date, more than 40 DAACPs have been detected, yet a very small number of peptide isomerases have been characterized and fewer still have been identified with a knowledge of a primary sequence. Recently DAACPs have been detected in the nervous system of a marine mollusk, Aplysia californica. The presence of such DAACPs had led to the hypothesis that an L/D peptide isomerase must exist in the nervous system of Aplysia, and an assay has been developed to partially purify and characterize the isomerase activity. A list of isomerase candidate proteins has been obtained through proteomics studies of partially purified CNS extracts. These proteins were further analyzed through single cell transcriptomics, and a system was developed for large-scale recombinant expression. Isomerase activity has also been detected in rodent systems on a non-endogenous synthetic peptide, yet while the activity suggests the presence of endogenous DAACPs, they have not yet been confirmed to exist in converted form. An assay has also been developed to characterize the activity in rodent systems, and a putative endogenous substrate has been identified. Furthermore, a system for activity guided purification was developed to obtain partially pure protein fractions, which were then subjected to proteomics analysis for candidate proteins. In addition to being critical for elucidating the biosynthetic pathway for DAACP production, the discovery of novel peptide isomerases will aid in a search for similar enzymes though sequence similarity and substrate specificity. In turn, investigations of tissue/cell type specific expression of these enzymes will help elucidate the functionality behind the isomerized peptides, as changes in expression pattern may reflect an unmet physiological need for the DAACP. Finally, the search for a novel free D-serine (D-Ser) racemase within the nematode Caenorhabditis elegans is described. While the endogenous conversion of L-Ser to D-Ser in C. elegans is conducted through a known serine racemase enzyme, the presence of D-Ser in racemase knockout models indicates multiple mechanisms for the synthesis of D-Ser. An investigation into the possible sources of D-Ser in C. elegans reveals the possibility of multiple enzymatic sources of D-Ser. The findings suggest further investigation is warranted to study the functional context of these novel serine processing enzymes.

Graduation Semester

2023-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Harvey M. Andersen IV

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