REQUERIMIENTOS DEL SERVICIO A CONTRATAR

In a physiological context, the acetylation of a lysine residue with an unfavorable sequence context for deacetylation (such as K38 of Ran) would be relatively sta- ble, which could result in a lasting e↵ect on protein function and/or intracellular signaling processes. The acetylation of a neighboring lysine could then lead to a release from this situation by stimulating the deacetylation of both residues by Sirtuins 1-3.

Results 89 Along these lines, the literature was screened for proteins, which have been shown to be acetylated at two neighboring lysine residues and deacetylated by SIRT1, -2 or -3. One such case is phosphoenolpyruvate carboxykinase 1 (PEPCK1), which plays a major role in gluconeogenesis and for which acetylation of residues K70, K71 and K594 has been reported. Acetylation of PEPCK1 promotes its degra- dation and is negatively regulated by SIRT2 (Jiang et al., 2011). Not only is PEPCK1 acetylated at two neighboring lysine residues but these two lysines are followed by a tyrosine (KKY) as is also case for RanAcK37. It appeared possible that this sequence represents a short motif for di-deacetylation. Interestingly, in an in vitro selection screen for cyclic peptidic inhibitors of SIRT2, 10 out of 15 iso- lated high affinity clones were found to contain the sequence R(I/V)(TFAcK)RY. The IC50’s of these cyclic peptides was in the low nanomolar range but also a

shorter linear peptide with the sequence RI(TFAcK)RY showed an IC50 of 31 nM

(Morimoto et al., 2012). Given that arginine is physicochemically similar to ly- sine, these results seemed to support the assumption that KKY is a target motif of SIRT2.

Full-length PEPCK1-WT, -AcK70, -AcK71, -AcK70/71 and, for comparison, also -AcK594 were purified with the GCEC. The resulting protein was approximately 80% pure and, when probed with the anti-AcK-AB, the acetylated variants showed a signal while PEPCK1-WT did not (Fig. 3.22a). As an additional measure of protein quality, the enzymatic activity of the recombinant PEPCK1 was confirmed. In this activity assay, the PEPCK1-catalyzed reaction of phosphoenolpyruvate to oxalacetate is coupled with the quantitative reduction of oxalacetate to malate by malate dehydrogenase. In the latter reaction step, NADH is oxidized to NAD+, which can be traced by a drop in emission at 470 nm when excited at 350 nm (see Material and Methods 2.3.14). The specificity of the assay was tested by sequen- tial addition of reaction components. A significant drop in fluorescence was only observed when PEPCK1 was added and the reaction was drastically accelerated upon addition of Mn2+ _(MnCl

2), which is a PEPCK1 co-factor. The slow reaction

rate observed without Mn2+ _{suggests that only a fraction of PEPCK1 protein pu-}

rified from E. coli is Mn2+_{-bound. All PEPCK1 variants displayed robust activity}

in this assay showing that the PEPCK1 was purified from E. coli in an active form. Moreover, PEPCK1-AcK71 showed an increased catalytic rate and reached a higher final fluorescence level. However, this observation has to be taken with caution since the purity between the di↵erent PEPCK1 varies and thus the true amount of enzyme in the assay cannot be accurately determined (Fig. 3.22b).

Results 90 Surprisingly, none of the acetylated PEPCK1-variants were deacetylated by SIRT2 in vitro when tested with low amounts of enzyme in preliminary experiments (not shown). To rule out that deacetylation just occurs at a much slower rate (com- pared for instance to RanAcK37), PEPCK1 was incubated for 2 h at 23 C with increasing amounts of SIRT2 up to an equimolar ratio. Again, under the experi- mental conditions used, no deacetylation was observed (Fig. 3.22c). Since only a fraction of the purified PEPCK1 was active due to the lack of the co-factor Mn2+_,

the experiment was repeated in MnCl2-containing bu↵er. Moreover, the concen-

tration of PEPCK1 was lowered to avoid possible multimerization or aggregation of PEPCK1, which could limit access of SIRT2 to the PEPCK1 acetylation sites. However, no deacetylation was observed at a 1:1 enzyme:substrate-ratio over the course of 2 h (Fig. 3.22d).

It was thus tested if mutation of the amino acid residues surrounding the di- acetylated site K70/71 would facilitate deacetylation by SIRT2. Two mutant vari- ants of PEPCK1 were cloned, each coding for three amino acid substitutions N- or C-terminally of the assumed binding motif KKY by the according Ran sequence (natural PEPCK1 sequence: RRLKKYDNC, natural Ran sequence: EFEKKY- VAT, ‘EFE’-mutant: EFEKKYDNC, ‘VAT’-mutant: RRLKKYVAT). However, in contrast to the EFE-mutant, the VAT-mutant could not be purified from E. coli de- spite significant e↵orts. The EFE-mutant was tested for deacetylation by SIRT2 in the di-acetylated PEPCK1-AcK70/71-background. Strikingly, as the time course experiment in Fig. 3.22e demonstrates, the PEPCK1-EFE mutant was deacety- lated efficiently with a similar rate compared to RanAcK37. Since the deacety- lation signal completely disappears over the course of the experiment, it is likely that both acetyl-moieties of PEPCK-70/71-EFE are removed by SIRT2. Taken together, these results suggest that, at least in vitro, PEPCK1 is not a substrate of SIRT2. However, mutation of three amino acids N-terminal to the assumed SIRT2 binding motif KKY is sufficient to convert PEPCK1 into a SIRT2-substrate, fa- cilitating deacetylation of both AcK70 and AcK71. Thus, these residues (and possibly also the residues VAT, which are found C-terminally of the KKY motif in Ran) appear to dictate the substrate binding of SIRT2.

Results 91