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  • Using the same techniques we found

    2022-12-02

    Using the same techniques, we found that UCP3 also has a half-life of between 1 and 4h [117]. In Diperodon HCl to UCP2 and UCP3, UCP1 and ANT had much longer half-lives and could not be degraded in the cell-free reconstituted system, suggesting their degradation is not mediated by the cytosolic proteasome [117]. We postulate that this fast turnover allows for rapid variations in UCP2 [87] and UCP3 levels in response to changes in nutrient fluxes, and the proteolytic pathway via the proteasome may allow the rapid regulation of these proteins in concert with other proteins involved in the same pathways. For example, in the pancreatic β-cell, the ubiquitin-proteasome system is responsible for regulating levels of other members of the insulin secretion pathway [118], [119], [120]. It is interesting to note that UCP1 protein levels are regulated by modulation of the synthesis and degradation in a concerted fashion [121]. The question arises as to whether UCP2 and UCP3 levels are also controlled in a concerted manner. Giardina et al. suggested that ROS not only increase UCP2 transcription but may also slow degradation [83]. However, we showed that the latter is in fact a confounding observation because only bioenergetic manipulations that increase ROS and simultaneously dissipate ATP and mitochondrial membrane potential result in slowing of UCP2 turnover [116]. This is not entirely unexpected since the proteasome-mediated degradation is ATP-dependent. Other than manipulation of ATP and mitochondrial membrane potential, to date, we have yet to find conditions that affect the rate of UCP2 degradation. Interestingly, the literature suggests that cellular proteolysis via the proteasome increases under catabolic conditions [122], which promote upregulation of UCP2 and UCP3. As such, UCP2 and UCP3 may be subject to constant rapid turnover, with variable expression being dependent primarily on rates of synthesis. It would be noteworthy to further examine whether regulators of UCP2 transcription or translation can also influence turnover, as this remains an alternative possibility.
    Concluding remarks
    Acknowledgements
    Introduction Protein microarrays can be divided into two general categories: ‘protein function microarrays’ and ‘protein-detecting microarrays’ (Figure 1) [1]. Protein function microarrays comprise purified proteins, protein domains, or peptides, and are generally used to study molecular recognition or to screen for putative interaction partners. In contrast, protein-detecting microarrays rely on reagents that recognize proteins in a selective fashion (e.g. antibodies) and are used to quantify the abundances and post-translational modification states of proteins in complex mixtures (e.g. cell lysates, tumor biopsies, and serum). The following discussion will address each of these categories separately.
    Protein function microarrays One of the primary goals of functional proteomics is to understand molecular recognition within the context of the proteome. Protein function microarrays provide a powerful way to assess binding selectivity across entire families of related proteins and, in the limit, across entire proteomes. In 2000, MacBeath and Schreiber showed that purified, recombinant proteins could be microarrayed on chemically derivatized glass substrates in a way that preserves their function [2]. Since then, variations of this technology have been used to study large collections of recombinant proteins. One approach Diperodon HCl that has been pursued extensively is to focus on families of interaction domains.
    Protein-detecting microarrays As detailed above, protein function microarrays enable broad and unbiased investigations of molecular recognition. Information gained from these studies can be used to map out biophysical and biochemical connections between proteins. To determine how information flows through these networks in a dynamic fashion, however, requires methods to measure the abundances and post-translational modification states of many different proteins in biological samples in high throughput. Protein-detecting microarrays provide this capability (Figure 1c,d). Since a large number of selective antibodies are commercially available, most studies in this area have relied on antibodies, in conjunction with microarray technology, to profile cellular lysates, tumor biopsies, and human serum.