Identification of sirtuin and its target as the ribosomal protein S4 in <i>Lactobacillus paracasei</i>

Abstract

Sirtuin is a protein with an enzymatic activity of NAD + -dependent protein deacetylation. It was first identified in yeast and its homologous genes have been widely found in various organisms. In bacteria, sirtuin gene was first described as cobB, encoding a cobalamin processing enzyme; and later its potential involvement in regulating acetylation levels of metabolic enzymes, transcription factors, chemotactic proteins and others have been reported. In order to study its physiological relevance in probiotic lactic acid bacteria, we analyzed the whole genome of three L. paracasei strains. All strains tested had sirtuin homolog genes designated hereby as sirA, and one of them had an additional gene designated as sirB. Following confirmation of their coding sequences by individual gene cloning, corresponding recombinant proteins have been generated and purified. The enzymatic characterization revealed that the intrinsic NAD + -dependent deacetylation activity of LpSirA (protein encoded by sirA) is comparable to human SIRT1. Furthermore, by blocking sirtuin activity using nicotinamide in vivo, together with an in vitro deacetylation reaction using recombinant LpSirA, we identified one of the target proteins in the lactic acid bacteria as the 30S ribosomal protein S4 (rpsD product).

Intracellular localization of sirtuin and cell length analysis of Lactobacillus paracasei suggest possible role of sirtuin in cell division and cell shape regulation

“…Previous finding in L. paracasei BL23 showed that a 28 kDa protein had increased its acetylation level with treatment of nicotinamide, a known sirtuin inhibitor in vivo, and had decreased its acetylation level upon supplementation of purified recombinant LpSirA with NAD + in vitro, indicating this protein as LpSirA target. This previous study also revealed one of the sirtuin target proteins in this cell was 30S ribosomal protein S4, suggesting role of LpSirA in protein synthesis [14]. Another study of note is the global acetylome analysis of E. coli proteins which showed that CobB mutation affected the acetylation levels of numerous proteins including those involved in protein synthesis (highly statistically significantly shorter and longer in comparison with the wild-type.…”

“…Cells were washed twice with PBS containing 0.1% (w/v) Tween 20 and blocked in PBS containing 2% (w/v) BSA, 0.3% (w/v) Triton X-100 and 0.1% (w/v) NaN 3 for 10 min. Cells were probed with a rabbit anti-LpSirA antibody [14] in PBS containing 2% (w/v) BSA, 0.3% (w/v) Triton X-100 and 0.1% (w/v) NaN 3 (1:250 dilution) for 1 h at 37 °C. Following four washes with PBS containing 0.1% (w/v) Tween 20, cells…”

“…The sirA gene, previously annotated as npd (NAD + -dependent protein deacetylase), was cloned and the enzymatic activity of its gene product (purified recombinant LpSirA) was demonstrated to be equivalent to human SIRT1 protein when using acetylated peptide substrate derived from p53. Furthermore, one of its endogenous substrates was identified as 30S ribosomal protein S4, indicating its role in protein synthesis in this bacterium [14].…”

“…Lactobacillus paracasei is one of the probiotic bacteria used widely in yoghurt and fermented milk products. This bacterium has single sirtuin homolog gene, sirA, in its genome together with no other Zn + -dependent protein deacetylase [14]. The sirA gene, previously annotated as npd (NAD + -dependent protein deacetylase), was cloned and the enzymatic activity of its gene product (purified recombinant LpSirA) was demonstrated to be equivalent to human SIRT1 protein when using acetylated peptide substrate derived from p53.…”

Bacterial Sirtuins Overview: An Open Niche to Explore

“…Some examples are the proteins Ku and LigD, components of the non-homologous end-joining (NHEJ) system from M. smegmatis and M. tuberculosis or the E. coli RNAse II (Li et al, 2011;Song et al, 2016). Finally, sirtuins have also been related to translation through E. coli alanyl-tRNA synthetase and Lactobacillus paracasei ribosomal S4 30S protein deacetylation (Atarashi et al, 2016;Torres-Barredo et al, 2018;Umehara et al, 2018). The elongation factor Tu from B. subtilis, TufA, is also regulated by reversible acetylation.…”