Small ubiquitin-related modifier (SUMO)-1 promotes glycolysis in hypoxia.

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress.

Analysis of 3-(acetylamino)-6-aminoacridine-derivatized oligosaccharides from recombinant monoclonal antibodies by liquid chromatography-mass spectrometry.

Glycosylation has been established as playing a pivotal role in various aspects of recombinant monoclonal antibodies (MAbs), ranging from pharmacokinetics to enhancement of effector function. Consequently, characterization of these oligosaccharides is of great importance and requires sensitive analytical techniques. Here we present a method for the rapid elucidation of 3-(acetylamino)-6-aminoacridine-labeled N-glycans present on MAbs using liquid chromatography-mass spectrometry. The technique uses the benefits of ultra-performance liquid chromatography systems in conjunction with small-particle-size amide columns capable of generating a fluorescence glycan profile of a MAb in 30 min, reducing the current run time by a factor of 6. The method is also compatible with online electrospray mass spectrometry, permitting the identification of glycans present. Overall, this strategy allows the confident determination of N-glycans present on recombinant MAbs in a significantly reduced amount of time.

Use of proteomics for the discovery of early markers of drug toxicity.

Toxicity and safety issues remain a significant problem for drug development efforts by pharmaceutical and biotechnology companies. Exisiting early biomarkers of toxicity are insufficient and this is demonstrated by the high failure rate of candidate therapeutics due to toxicity problems. It is anticipated that the advent of 'omic' technologies should facilitate a comprehensive understanding of the perturbation of biological systems by toxic insults and, as such, will lead to better predictive models of toxicity for use in drug development. The field of proteomics continues to develop rapidly and it is already evident that proteomic approaches have much to contribute to the field of 'systems toxicology' and to the development of novel biomarkers of toxicity. Here, the key proteomic approaches are reviewed, their applications in pharmaceutical toxicology are described and what shape future developments in this arena might take is considered.