The critical role of S-lactoylglutathione formation during methylglyoxal detoxification in Escherichia coli.

Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB.

Base excision repair activities required for yeast to attain a full chronological life span.

The chronological life span of yeast, the survival of stationary (G0) cells over time, provides a model for investigating certain of the factors that may influence the aging of non-dividing cells and tissues in higher organisms. This study measured the effects of defined defects in the base excision repair (BER) system for DNA repair on this life span. Stationary yeast survives longer when it is pre-grown on respiratory, as compared to fermentative (glucose), media. It is also less susceptible to viability loss as the result of defects in DNA glycosylase/AP lyases (Ogg1p, Ntg1p, Ntg2p), apurinic/apyrimidinic (AP) endonucleases (Apn1p, Apn2p) and monofunctional DNA glycosylase (Mag1p). Whereas single BER glycosylase/AP lyase defects exerted little influence over such optimized G0 survival, this survival was severely shortened with the loss of two or more such enzymes. Equally, the apn1delta and apn2delta single gene deletes survived as well as the wild type, whereas a apn1delta apn2delta double mutant totally lacking in any AP endonuclease activity survived poorly. Both this shortened G0 survival and the enhanced mutagenicity of apn1delta apn2delta cells were however rescued by the over-expression of either Apn1p or Apn2p. The results highlight the vital importance of BER in the prevention of mutation accumulation and the attainment of the full yeast chronological life span. They also reveal an appreciable overlap in the G0 maintenance functions of the different BER DNA glycosylases and AP endonucleases.

A yeast-based assay reveals a functional defect of the Q488H polymorphism in human Hsp90alpha.

It has been argued that the molecular chaperone Hsp90 guards the organism against genetic variations by stabilizing variant Hsp90 substrate proteins. However, little is known about polymorphisms affecting its own functions. We have followed up on a recent study describing two polymorphisms that alter the amino acid sequences of the two Hsp90 isoforms Hsp90alpha and Hsp90beta. Hsp90 is essential for cell proliferation in the budding yeast Saccharomyces cerevisiae, but the human proteins can replace the endogenous ones. In this growth assay, the variant V656M of Hsp90beta was indistinguishable from wild-type. In contrast, the Hsp90alpha variant Q488H, which carries an alteration of a very highly conserved residue, was severely defective for growth compared to wild-type Hsp90alpha. Hence, the characteristics of this yeast-based system-simplicity, rapidity, low cost-make it ideal for phenotype screening of polymorphisms in HSP90 and possibly many other human genes.