A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors.

Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.

Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol.

BACKGROUND: In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol. METHODS: Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol. RESULTS: In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol. CONCLUSION: This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol. GENERAL SIGNIFICANCE: The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.

Structural determinants of metal specificity in the zinc transport protein ZnuA from synechocystis 6803.

A number of bacterial metal transporters belong to the cluster 9 family of ABC transporters. The residues in the periplasmic domain thought to be involved in metal binding seem highly conserved and yet the transporters have varying metal specificity. To solve this seeming paradox and ascertain how metal specificity is exacted, the structure of ZnuA, the periplasmic domain of a zinc transporter from Synechocystis 6803, has been determined to a resolution of 1.9A. In previously determined structures of homologous proteins, four residues chelate the bound metal. From sequence alignments of the cluster 9 metal transporters, the fourth residue in this metal-binding site, an aspartate, is also present in the appropriate position in the ZnuA sequence. However, this result is misleading, since our structural data indicate that zinc binds via only three histidine residues and the aspartate is replaced by a large hydrophobic cavity. We propose that ZnuA binds zinc over manganese by providing only three ligating residues. ZnuA has a highly charged and mobile loop that protrudes from the protein in the vicinity of the metal-binding site. Similar loops are found in other types of zinc transporters but not manganese transporters. Therefore, we propose that the function of this domain is to act as a zinc chaperone to facilitate acquisition. Therefore, while Mn2+ transporters can bind Zn2+ in vitro they may not be able to acquire it in vivo without this structure because of the low concentration of free Zn2+.

In vitro evidence of inhibition of mitochondrial protease processing by HIV-1 protease inhibitors in yeast: a possible contribution to lipodystrophy syndrome.

Highly active antiretroviral therapy has been associated with the emergence of lipodystrophy syndromes that have clinical features commonly seen in patients with mitochondrial dysfunction. The effect of therapeutic protease inhibitors (PIs) on mitochondrial function is unknown. Mitochondrial matrix space proteins possess an amino-terminal leader peptide that is removed by the mitochondrial processing protease (MPP). Lack of cleavage could result in non- or dysfunctional mitochondrial proteins. The effects of different PIs on protease processing using pure MPP or yeast mitochondria, recognized models for mammalian counterparts, were examined in vitro. Multiple PIs were found to inhibit MPP, evidenced by accumulation of immature pALDH and decreased levels of processed ALDH. Both indinavir and amprenavir at 5.0 mg/ml resulted in significant inhibition of MPP. Although inhibition of MPP was also observed with ritonavir and saquinavir, the inhibition was difficult to quantify due to background inhibition of MPP by DMSO that was required to solubilize the drugs for the in vitro studies. Indinavir was also shown to inhibit MPP within yeast mitochondria. Lack of processing may impair mitochondrial function and contribute to the observed mitochondrial dysfunctions in patients receiving HAART and implicated in antiretroviral-associated lipodystrophy.

Chemical modifications to study mutations that affect the ability of the general base (E268) to function in human liver mitochondrial aldehyde dehydrogenase.

The action of a general base is needed in two possible steps during the aldehyde dehydrogenase catalyzed oxidation of an aldehyde to an acid. The base is glutamate at position 268 in the cytosolic and mitochondrial class 1 and 2 enzyme. A chemical modification approach was undertaken to determine if the base were necessary in the initial attack of the nucleophilic cysteine (302) on the aldehyde as well as the attack by water on the acyl intermediate formed after the aldehyde is oxidized. A metabolite of disulfiram, S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), was used as the modifying agent. Three recombinantly expressed mutant forms of the human mitochondrial enzyme along with the native one were used. These were the E268Q mutant that was lacking the general base; the E487K Oriental variant of the enzyme and R475Q, a mutant possessing the residue that binds to E487. As expected, the E268Q mutant was inactivated very slowly compared with the native or other mutants that were inactivated more slowly than the native enzyme. The presence of NAD did not increase the rate of inactivation except with the R475Q mutant. It is concluded that it is necessary to activate the cysteine at the active site to make it a good nucleophile as well to activate water during the hydrolysis of the thio-acyl intermediate. Further, it is surmised that the reason some mutants have a lowered specific activity is that in those the general base is not capable of functioning as it does in the native enzyme.

Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase.

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.

Possible regulatory role for the histidine-rich loop in the zinc transport protein, ZnuA.

A number of bacterial metal transporters belong to the ABC transporter family. To better understand the structural determinants of metal selectivity of one such transporter, we previously determined the structure of the periplasmic domain of a zinc transporter, ZnuA, from Synechocystis 6803 and found that ZnuA binds zinc via three histidines. Unique to these ABC zinc transporters, ZnuA has a highly charged and mobile loop that protrudes from the protein in the vicinity of the metal binding site that we had suggested might facilitate zinc acquisition. To further examine the function of this loop, the structure and zinc binding properties of two ZnuA variants were determined. When the loop is entirely deleted, zinc still binds to the three histidines. However, unlike what was suggested from the structure of a similar solute binding protein, TroA, release of zinc occurs concomitantly with large conformational changes in two of the three chelating histidines. These structural results combined with isothermal titration calorimetry data demonstrate that there are at least two classes of zinc binding sites: the high-affinity site in the cleft between the two domains and at least one additional site on the flexible loop. This loop has approximately 100-fold weaker affinity for zinc than the high-affinity zinc binding site, and its deletion does not affect the high-affinity site. From these results, we suggest that this region might be a sensor for high periplasmic levels of zinc.