Palmitoylation down PAT

Post-translational modifications decorate proteins with diverse chemical groups that confer new biochemical properties. For example, protein palmitoylation results from the thioester conjugation of palmitic acid to a cysteine residue in the target protein. Like other lipid-modified proteins, palmitoylated proteins become tethered to the cytoplasmic side of the cell membrane and are involved in processes such as cellular signaling and membrane trafficking. However, unlike other lipid modifications, palmitoylation has been shown to be reversible, and this seems to be important for its cellular effects. Despite the recent identification of protein acetyltransferase (PAT) enzymes, which synthesize palmitoylated proteins, little is known about which proteins are modified, the consensus sequences that mark palmitoylation sites or the PAT enzymes themselves. In a recent study, Davis and co-workers used a proteomics approach to identify palmitoylated proteins in Saccharomyces cerevisiae and discovered that a certain class of PAT enzymes, from the DHHC protein family, is largely responsible for palmitoylation in cells. The authors isolated and identified palmitoylated proteins from yeast protein extracts using proteomics tools, and they validated palmitoylation reactions of candidate proteins in cells. The approach revealed 12 of the 15 proteins known to be palmitoylated in yeast, and 35 additional palmitoylation targets, including SNAREs, G proteins, amino acid permeases and mannosyltransferases. Additional studies revealed consensus sequences for palmitoylation of transmembrane protein domains and identified members of the DHHC zinc finger protein–like family, which carry out most of the palmitoylation in cells. The current study provides an important new inventory of the palmitoylation machinery in yeast, which should lead to greater molecular-level understanding of this post-translational modification. (Cell 125, 1003–1013, 2006) TLS

Green in tooth and claw

Heme, a vital component in respiration, is an extremely potent prooxidant in its free form. To avoid toxicity, cells across several species tightly regulate its degradation to the green molecule biliverdin. However, for bloodsucking insects, which digest huge quantities of heme relative to their body mass, the normal degradation pathway may not be sufficient. Indeed, Oliveira and colleagues find that heme breakdown in the 'kissing bug' Rhodnius prolixus, a vector of Chagas disease, follows a unique pathway in which two hydrophilic groups are appended to the heme ring before oxidative cleavage. The authors injected female insects with 14C-labeled heme and analyzed the resultant tissue homogenates by HPLC. They isolated a molecule that had an absorption profile similar to that of biliverdin but was substantially more hydrophilic. ESI-MS revealed the molecule, dubbed RpBV, to be biliverdin coupled to two cysteine residues. The authors further demonstrated that the presence of these residues can be traced to attachment (and subsequent processing) of cysteinylglycine groups to the heme via thioether linkages. The purpose of these hydrophilic heme modifications may be to facilitate excretion after a blood meal. A more complete model of heme breakdown should provide insight into both the ways insects can control gut parasites and the ways we can control insects. (Proc. Natl. Acad. Sci. USA 103, 8030–8035, 2006) KM

An antitumor house of cards

C-1027, a small molecule–apoprotein complex, is a potent antitumor agent that cleaves DNA via radical-mediated hydrogen abstraction. The small chromophore consists of a reactive enediyne ring, which is responsible for cleavage of the DNA backbone, and a benzoxazine side chain, hydrolysis of which decreases DNA-binding affinity by 400-fold. C-1027 is unique among related compounds in that the enediyne component of the isolated chromophore spontaneously rearranges in solution, eradicating the chromophore's activity. Thus, the protein shell serves to protect the active form of the chromophore before delivery to the DNA target; however, the mechanistic details of this symbiosis are unresolved. It has previously been shown that the protein slows decomposition of the radical-generating enediyne moiety by providing only a poor hydrogen donor in the form of a nearby glycine. In the current paper by Inoue and colleagues, stability assays confirm this effect: when the availability of the hydrogen donor is further diminished by substitution with deuterated glycine, the lifetime of the active species is increased up to six-fold. However, when hydrogen abstraction within the complex does occur, the protein undergoes oxidative cleavage at the proximal glycine. The authors further demonstrate that the apoprotein protects the benzoxazine unit of the chromophore. Thus the chromophore, when compromised, mediates decomposition of the protein scaffold, which in turn allows hydrolysis of benzoxazine, the final reactive unit. The unique capability of this complex to stabilize the highly reactive form of a targeted drug provides new possibilities in the development of drug delivery systems. (J. Am. Chem. Soc., published online 24 May 2006, doi:10.1021/ja060724w10.1021/ja060724w) CG

PDI SNOed under

The accumulation of aberrant or misfolded proteins that leads to neurodegenerative disorders such as Parkinson disease is thought to reflect ongoing cell stress. Uehara, Lipton and colleagues strengthen this connection by studying the stress chaperone protein disulfide isomerase (PDI). PDI is involved in disulfide bond formation in the endoplasmic reticulum through the oxidation of newly synthesized proteins. Its upregulation helps protect neurons from the slow accumulation of misfolded aggregation-prone proteins. Now, Uehara, Lipton and colleagues show that PDI can be S-nitrosylated (forming SNO-PDI) and further oxidized by reactive oxygen species on cysteine residues of two distinct thioredoxin-like domains both in vitro and in vivo. The authors found SNO-PDI in a Parkinson disease cell model as well as in autopsies of diseased human brains. Functionally, SNO-PDI lacks the chaperone and isomerase activities of the wild-type PDI, suggesting that S-nitrosylation inhibits the neuroprotective capacity of PDI. Indeed, synphilin-1, a component of Lewy body inclusions in Parkinson neurons, could not be cleared by SNO-PDI, leading to the formation of these inclusions. In addition to its roles in linking free-radical stress and protein misfolding, PDI (but not SNO-PDI) helps mitigate the apoptotic responses to the accumulation of unfolded proteins and the unfolded protein response. These results suggest that by virtue of its chaperone activities, PDI protects neurons from degenerative damage and apoptosis, though this protection is diminished when PDI has been subjected to NO-induced insults. (Nature 441, 513–517, 2006) MB

Research Highlights written by Mirella Bucci, Catherine Goodman, Kaspar Mossman and Terry L. Sheppard