John J. Heikkila

Modest PGC-1α Overexpression in Muscle in Vivo Is Sufficient to Increase Insulin Sensitivity and Palmitate Oxidation in Subsarcolemmal, Not Intermyofibrillar, Mitochondria

PGC-1α overexpression in skeletal muscle, in vivo, has yielded disappointing and unexpected effects, including disrupted cellular integrity and insulin resistance. These unanticipated results may stem from an excessive PGC-1α overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1α overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1α overexpression selectively targeted subsarcolemmal (SS) mitochondrial proteins and fatty acid oxidation, because SS mitochondria are metabolically more plastic than intermyofibrillar (IMF) mitochondria. Among metabolically heterogeneous rat hindlimb muscles, PGC-1α was highly correlated with their oxidative fiber content and with substrate transport proteins (GLUT4, FABPpm, and FAT/CD36) and mitochondrial proteins (COXIV and mTFA) but not with insulin-signaling proteins (phosphatidylinositol 3-kinase, IRS-1, and Akt2), nor with 5′-AMP-activated protein kinase, α2 subunit, and HSL. Transfection of PGC-1α into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1α protein by 23-25%. This also induced the up-regulation of transport proteins (FAT/CD36, 35-195%; GLUT4, 20-32%) and 5′-AMP-activated protein kinase, α2 subunit (37-48%), but not other proteins (FABPpm, IRS-1, phosphatidylinositol 3-kinase, Akt2, and HSL). SS and IMF mitochondrial proteins were also up-regulated, including COXIV (15-75%), FAT/CD36 (17-30%), and mTFA (15-85%). PGC-1α overexpression also increased palmitate oxidation in SS (RTA, +116%; WTA, +40%) but not in IMF mitochondria, and increased insulin-stimulated phosphorylation of AKT2 (28-43%) and rates of glucose transport (RTA, +20%; WTA, +38%). Thus, in skeletal muscle in vivo, a modest PGC-1α overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.

Use of a Scanning Densitometer or an ELISA Plate Reader for Measurement of Nanogram Amounts of Protein in Crude Extracts from Biological Tissues

Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w/v, in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng.microliter-1, respectively.

Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity

Abstract Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C. An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.

Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster

Abstract The Drosophila melanogaster family of small heat shock proteins (sHsps) is composed of 4 main members (Hsp22, Hsp23, Hsp26, and Hsp27) that display distinct intracellular localization and specific developmental patterns of expression in the absence of stress. In an attempt to determine their function, we have examined whether these 4 proteins have chaperone-like activity using various chaperone assays. Heat-induced aggregation of citrate synthase was decreased from 100 to 17 arbitrary units in the presence of Hsp22 and Hsp27 at a 1:1 molar ratio of sHsp to citrate synthase. A 5 M excess of Hsp23 and Hsp26 was required to obtain the same efficiency with either citrate synthase or luciferase as substrate. In an in vitro refolding assay with reticulocyte lysate, more than 50% of luciferase activity was recovered when heat denaturation was performed in the presence of Hsp22, 40% with Hsp27, and 30% with Hsp23 or Hsp26. These differences in luciferase reactivation efficiency seemed related to the ability of sHsps to bind their substrate at 42°C, as revealed by sedimentation analysis of sHsp and luciferase on sucrose gradients. Therefore, the 4 main sHsps of Drosophila share the ability to prevent heat-induced protein aggregation and are able to maintain proteins in a refoldable state, although with different efficiencies. The functional reasons for their distinctive cell-specific pattern of expression could reflect the existence of defined substrates for each sHsp within the different intracellular compartments.

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup.

Heat shock protein gene expression during Xenopus development

Abstract. Stress-induced heat shock protein gene expression is developmentally regulated during early embryogen esis of the frog, Xenopus laevis. For example, a number of heat shock protein genes, such as hsp70,hsp90, and ubiquitin are not heat-inducible until after the midblastula stage of embryogenesis. Furthermore, the family of small heat shock protein genes, hsp30, are differentially expressed after the midblastula stage as well as being regulated at the level of mRNA stability. Many of these stress proteins are also synthesized constitutively during oogenesis and embryogenesis during which they may act as molecular chaperones as well as being involved in sequestering proteins in an inactive state until required by the developing embryo. Furthermore the induction of these stress protein genes has been correlated with enhanced thermoresistance. During stressful conditions heat shock proteins probably prevent aggregation or misfolding of damaged protei ns within the embryo.

Heat shock protein gene expression and function in amphibian model systems.

Heat shock proteins (HSPs) are molecular chaperones that are involved in protein folding and translocation. During heat shock, both constitutive and stress-inducible HSPs bind to and inhibit irreversible aggregation of denatured protein and facilitate their refolding once normal cellular conditions are re-established. Recent interest in HSPs has been propelled by their association with various human diseases. Amphibian model systems, as shown in this review, have had a significant impact on our understanding of hsp gene expression and function. Some amphibian hsp genes are expressed constitutively during oogenesis and embryogenesis, while others are developmentally regulated and enriched in selected tissues in a stress-inducible fashion. For example, while hsp70 genes are heat-inducible after the midblastula stage, hsp30 genes are not inducible until late neurula/early tailbud. This particular phenomenon is likely controlled by chromatin structure. Also, hsp genes are expressed during regeneration, primarily in response to wounding-associated trauma. The availability of amphibian cultured cells has enabled the analysis of hsp gene expression induced by different stresses (e.g. cadmium, arsenite, proteasome inhibitors etc.), HSP intracellular localization, and their involvement in stress resistance. Furthermore, hyperthermia treatment of adult amphibians reveals that certain tissues were more sensitive than others in terms of hsp gene expression. Finally, this review details the evidence available for the role of amphibian small HSPs as molecular chaperones.

Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos.

We employed whole-mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock-induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33 degrees C heatshock. The lowest temperature capable of inducing this pattern was 30 degrees C. Placement of embryos at 22 degrees C following a 1-h 33 degrees C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues.

Evaluation of stress-inducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis

In this study we have evaluated stress-inducible hsp90 mRNA accumulation as a potential molecular biomarker in Xenopus laevis. In order to obtain a probe for Northern blot analysis we employed a PCR-based approach using degenerate primers for the amplification and cloning of an hsp90 gene sequence from Xenopus laevis. The deduced amino acid sequence is 102 amino acids in length and exhibited the highest degree of identity with zebrafish and human hsp90 beta genes. Furthermore, the putative intron and exon boundaries of this fragment are the same as hsp90 beta in chicken, mouse and human, indicating that the fragment represents a Xenopus hsp90 beta-like gene. Northern blot analyses revealed that this gene was constitutively expressed in cultured A6 cells. While heat shock and sodium arsenite exposure resulted in the increased accumulation of hsp90 mRNA in A6 cells, treatment with cadmium chloride and zinc chloride did not. Also, exposure of A6 cells to concurrent heat shock and sodium arsenite produced a mild synergistic response with respect to hsp90 mRNA levels in contrast to hsp70 mRNA levels which displayed a strong synergistic effect. Finally, hsp90 mRNA was detected constitutively throughout early embryogenesis but was heat-inducible only in late blastula and later stages of development. Given the normal abundance and limited stress-induced accumulation of hsp90 mRNA, it may not have a great deal of potential as a molecular biomarker compared to hsp70 and hsp30 mRNA. However, it may be useful in conjunction with other stress protein mRNAs to establish a set of biomarker profiles to characterize the cellular response to a stressful or toxic agent.

Comparison of regulatory and structural regions of the Xenopus laevis small heat-shock protein-encoding gene family

We have isolated several unique Xenopus laevis hsp30 (encoding heat-shock protein 30) genomic clones, one of which contains two complete hsp30 genes (hsp30C and hsp30D), as well as the promoter and N-terminal coding region of a third gene (hsp30E). Nucleotide sequence and restriction enzyme analysis revealed that this gene cluster is different from a cluster isolated previously. The hsp30C and hsp30D genes encode proteins of approx. 24 kDa. In all, the hsp30 gene family contains a minimum of seven genes. The strand exchange and breakage of the duplication events which generated this gene family appear to have occurred within tracts of DNA which potentially can assume a Z-DNA conformation. Comparing the amino acid (aa) sequences of each known Hsp30 protein with bovine alpha-crystallin revealed a high degree of shared conservation of aa that constitute the major structural feature(s) of alpha-crystallin.