Climate change and associated increases in water temperatures may impact physiological performance in ectotherms and exacerbate endangered species declines. We used an integrative approach to assess the impact of elevated water temperature on two fishes of immediate conservation concern in a large estuary system, the threatened longfin smelt (Spirinchus thaleichthys) and endangered delta smelt (Hypomesus transpacificus). Abundances have reached record lows in California, USA, and these populations are at imminent risk of extirpation. California is currently impacted by a severe drought, resulting in high water temperatures, conditions that will become more common as a result of climate change. We exposed fish to environmentally relevant temperatures (14°C and 20°C) and used RNA sequencing to examine the transcriptome-wide responses to elevated water temperature in both species. Consistent with having a lower temperature tolerance, longfin smelt exhibited a pronounced cellular stress response, with an upregulation of heat shock proteins, after exposure to 20°C that was not observed in delta smelt. We detected an increase in metabolic rate in delta smelt at 20°C and increased expression of genes involved in metabolic processes and protein synthesis, patterns not observed in longfin smelt. Through examination of responses across multiple levels of biological organization, and by linking these responses to habitat distributions in the wild, we demonstrate that longfin smelt may be more susceptible than delta smelt to increases in temperatures, and they have little room to tolerate future warming in California. Understanding the species-specific physiological responses of sensitive species to environmental stressors is crucial for conservation efforts and managing aquatic systems globally.

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.

Three sets of isozymes of glutathione-S-transferase (GST) have been identified in human tissues. They differ in their tissue distribution, incidence of genetic variation, susceptibility to inactivation by N-ethylmaleimide and in their electrophoretic mobilities. The GST1 isozymes exhibit four phenotypes, including a common 'null' phenotype attributable to different combinations of three autosomal alleles GST1 1, GST1 2 and GST1 0 of frequency 0.13, 0.23 and 0.64, respectively, in the European population. The genetic polymorphism of GST1 is easily demonstrable in adult liver, kidney, adrenal and stomach but the isozymes are only weakly expressed in skeletal and cardiac muscle and not at all in fetal liver, fibroblasts, erythrocytes, lymphocytes and platelets. The GST2 isozymes also exhibit variant patterns but these are probably due to post-synthetic modification rather than allelic variation. The GST2 isozymes are not detectable in erythrocytes, platelets, cultured fibroblasts or lymphocytoid cells but are found in many other tissues, including fetal liver. GST3 isozymes were found as relatively strong components in every tissue examined except adult liver, with slight tissue to tissue variability in electrophoretic mobility.

Salmonids are particularly susceptible to warming and hypoxia during development in redds. We reared Chinook salmon embryos and alevins under chronic warming and hypoxia to evaluate the effects of each stressor individually and their interaction. Warming and hypoxia affected survival, physiological performance and development with management implications for salmon conservation.

The expression of the GST1, GST2, and GST3 loci in fetal, neonatal, and infant tissues has been studied using starch gel electrophoresis and chromatofocusing. Each locus demonstrated developmental changes in expression, some of which were specific to a single tissue while others occurred in several tissues. GST1 was not usually expressed in any of the tissues studied before 30 weeks of gestation but steadily increased thereafter until adult levels were reached in late infancy. In neonates and older infants the frequencies of the GST1*0, GST1*1, and GST1*2 alleles were 0.79, 0.07, and 0.14, respectively. GST2 was always expressed in liver and adrenal but was only weakly expressed in spleen, cardiac muscle, and diaphragm. In kidney this locus was not usually expressed until nearly 1 year after birth. The GST3 isoenzymes were present in all fetal, neonatal, and infant tissues, although their expression in liver decreased after 30 weeks of gestation. Other isoenzymes with fast anodal mobilities were also identified in several tissues; these are believed to be GST3 isoenzymes that have undergone posttranslational modification rather than products of the putative GST4 locus. No specifically fetal isoenzymes were detected.