The Chaperone-Like Protein HYPK Acts Together with NatA in Cotranslational N-Terminal Acetylation and Prevention of Huntingtin Aggregation

Abstract

The human NatA protein N ␣ -terminal-acetyltransferase complex is responsible for cotranslational Nterminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry, we identified endogenous HYPK, a Huntingtin (Htt)-interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p, and hNaa15p were associated with polysome fractions, indicating a function of HYPK associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels, possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G 0 /G 1 phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-enhanced green fluorescent protein (Htt-EGFP) fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the data indicate that the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and for N-terminal acetylation.

An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation

“…Generally, the incubation time for His 6 -Ssp-Httex1-43Q was limited to 6 h to prevent aggregation of the splicing product, Httex1-43Q. Splicing products were separated by preparative C4 RP-HPLC, yielding Httex1 with Ͼ95% purity based on analysis of purified fractions by RP-UHPLC The Httex1 proteins are devoid of the first methionine, as it was reported to be cleaved off in vivo (48). C, the expression of His 6 -Ssp-Httex1-23Q/43Q (arrow) was analyzed by SDS-PAGE.…”

N-terminal acetylation promotes synaptonemal complex assembly in C. elegans

“…Mutations in NATs are associated with various phenotypes in different organisms, including in humans, where mutations in NAA10, the catalytic subunit of NatA, lead to a global developmental delay and Ogden and Lenz microphthalmia syndromes (Rope et al 2011;Esmailpour et al 2014;Myklebust et al 2015;Popp et al 2015). Despite its abundance, the molecular functions of this modification have been characterized for only a handful of cases, where N-terminal acetylation can affect protein targeting to membranes, protein-protein interactions, protein folding, or protein degradation (Urbancikova and Hitchcock-DeGregori 1994;Setty et al 2004;Arnesen et al 2010;Hwang et al 2010;Scott et al 2011;Holmes et al 2014). However, the extent to which these specific functions operate in the proteome remains unclear.…”

Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

“…Recently, human Naa50p (hNaa50p) was reported to display lysine or N -acetyltransferase as well as NAT activity (16), the latter was defined as NatE activity (16). Interestingly, the chaperone-like, Huntingtin interacting protein HYPK, identified as a novel stable interactor of human NatA, was functionally implicated in the N-terminal acetylation of an in vivo NatA substrate, demonstrating that NAT complex formation and composition may have an overall influence on the observed (degree of) N ␣ -acetylation (17). Further, subunits of the human NatA complex have been coupled to cancer-related processes and differentiation, with altered subunit expression reported in papillary thyroid carcinoma, neuroblastoma, and retinoic acid induced differentiation.…”