Diverse functions of LanC-like proteins in mammalian cells

Bacterial LanC proteins have been extensively studied and shown to be responsible for installing thioether crosslinks in precursors of lanthipeptides. These modifications allow lanthipeptides to possess different biological activities such as antimicrobial activity. LanC-like proteins have been identified in various eukaryotic cells, but their function is not known. Intriguingly, the structure of the bi-functional enzyme CylM was solved and the dehydratase domain of LanM unexpectedly showed structural homology with mammalian kinases such as the mammalian target of rapamycin (mTOR). This finding has led us to look for potential interaction partners for LanCL proteins. In Chapter 2, I demonstrate that LanCL proteins physically interact with many cellular kinases in mammalian cells by using pulldown assays. Since LanCL proteins have been shown to bind glutathione (GSH), I hypothesized that LanCL proteins might act as post-translational modification enzymes to modify proteins/peptides. In this study, dehydroamino acids containing peptides that mimic the activation loop of Erk and Akt protein kinases were synthesized. LanCL proteins were shown to add GSH to these dehydroamino acids containing peptides. The same C-glutathionylation was also observed by using dehydroamino acid containing proteins as substrates. These data show that LanCL proteins have flexible capabilities to catalyze C-glutathionylation on different substrates. GC-MS analysis showed that the C-glutathionylation catalyzed by LanCL2 involves formation of DL-(methyl)lanthionine linkages. In order to probe the biological function of C-glutathionylation in mammalian cells, affinity purification mass spectrometry-based (AP-MS) proteomic analysis was performed which is presented in Chapter 3. Based on the bioinformatic analysis of the proteomic data, it seems LanCL2 participates in different metabolic pathways as well as protein degradation pathways. I therefore hypothesized that C-glutathionylation might act as a beacon for protein degradation; however, my preliminary data do not support a model that C-glutathionylation is a tag for protein degradation in mammalian cells. Instead, LanCL proteins may function to remove detrimental electrophilic sites from the proteome. In addition to acting as an enzyme in mammalian cells, we showed that LanCL2 might also act as a positive regulator for PPAR activity in Chapter 4. LanCL2 knockdown (KD) in preadipocytes led to disruption of their differentiation capability and pulldown assays showed that LanCL2 physically interacts with PPAR in HEK293 and 3T3-L1 cells. An in vitro binding study further showed that LanCL2 directly interacts with PPAR. Finally, luciferase reporter assays showed that LanCL2 is important for PPAR transactivation activity. Taken together, my thesis research suggests different roles of LanCL proteins in mammalian cells.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2020

Author dc:creator

• Lai, Kuan-Yu Nick

Contributors dc:contributor

• van der Donk, Wilfred A.
• Nair, Satish K.
• Procko, Erik
• Chen, Jie

• LanCL, Enzyme, Glutathionylation

Statement dc:rights

• Copyright 2019 Kuan-Yu Nick Lai

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/108214

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/108214