An enhanced recombinant amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affecting α-synuclein oligomerisation

Abstract

Amino-terminal acetylation is a ubiquitous protein modification affecting the majority of eukaryote proteins to regulate stability and function. We describe an optimised recombinant expression system for rapid production of amino terminal-acetylated proteins within bacteria. We go on to describe the system's use in a fluorescence based in vivo assay for use in the high-throughput screen to identify drugs that impact amino-terminal acetylation-dependent oligomerisation. These new tools and protocols will allow researchers to enhance routine recombinant protein production and identify new molecules for use in research and clinical applications.

Keywords: Nat; Parkinson's disease; Tropomyosin; amino-α-acetyl-transferase; α-synuclein.

Figure 1

Bacterial Nt‐acetylation expression system. (A) Schematic outline of Nat constructs containing genes encoding catalytic and regulatory subunits of the each Nt‐acetylation complex under the control of T7 promoters. Expression of the substrate from a separate construct is under the control of the rhamnose promoter. (B) Cell lysates from BL21(DE3) cells containing either pRham‐Tm4.2 (left), pRham‐Tm4.2 and pNatA (middle), or pRham‐αSkTm and pNatB (right) were separated by SDS/PAGE following sequential addition of IPTG and Rhamnose, and visualised using coomassie stain. * and ** denote bands corresponding to Nat regulatory and catalytic subunits respectively. (C) Summary of amino‐termini tested and Nt‐acetylation efficiency. Culturing cells at lower temperatures as well as reducing the strength of target protein induction (by adding reduced quantities of IPTG/rhamnose) also resulted in improved Nt‐acetylation efficiency. We have observed 100% Nt‐acetylation of a number of target proteins (e.g. Tpm, Calmodulin) when cells are cultured in minimal media (e.g. for ¹⁵N labelling of proteins), however, isotope‐enriched rich media (NZY) can improve Nt‐acetylation in some cases.

Figure 2

Bacterial Nt‐acetylation systems do not inhibit cell growth or production of target substrate proteins. (A) Growth curves generated from averages from 10 replicate cultures of BL21(DE3) E. coli cells containing an empty pACYCDUET vector (dashed lines and empty circles), pNatA (grey lines & grey filled circles) or pNatB (black lines and black filled circles), all grown on the same 96‐well plate. Expression of NatA or NatB does not impact bacterial growth. (B) Cerulean fluorescence signal from cultures of BL21(DE3) cells contained pJC20‐cerulean3‐cdc8 (triangles), pJC20‐cdc8‐cerulean3 (diamonds) pJC20‐αSyn‐cerulean3 (circles) together with either pNatA (empty shape) or pNatB (filled shape). Expression of NatA or NatB does not impact expression of coexpressed recombinant proteins. (C) Yellow fluorescence signal from BL21(DE3) cells containing pET‐BiFC together with either pNatA (empty square) or pNatB (filled squares) illustrate Nt‐acetylation does not impact BiFC fluorescence.

Figure 3

An in vivo BiFC system to identify Nt‐acetylation state‐specific αSyn oligomerisation. (A) Growth (empty circles) and fluorescence (filled circles) curves of BL21(DE3) cells containing pET‐αSyn‐BiFC together with either pNatA (grey shapes) or pNatB (black shapes) show while αSyn Nt‐acetylation does not impact cell growth (OD ₅₉₅), it affects BiFC signal from αSyn oligomers (Au). (B) αSyn‐BiFC fluorescence (upper panels, green bottom panels) and Congo Red staining (middle panels, magenta bottom panels) of BL21(DE3) cells containing pET‐αSyn‐BiFC with either pNatA (left panels) or pNatB (right panels) indicate Nt‐acetylation promotes αSyn amyloid formation in bacteria. (C) Fluorescence (filled circles) curves of BL21(DE3) cells containing pET‐αSyn‐BiFC and pNatB grown in the presence of DMSO (empty circles), 5 mm Gefitinib (filled diamonds), 5 mm Imatinib (filled circles) or 5 mm Lapatinib (filled triangles) illustrate this class of tyrosine kinase inhibitors promote formation of amyloids of Nt‐acetylated αSyn.