Yanmei Zhao

Lifespan extension and elevated hsp gene expression in Drosophila caused by histone deacetylase inhibitors

SUMMARY The heat shock proteins (Hsps) play a positive role in lifespan determination, and histone acetylation has been shown to be involved in transcription of hsp genes in Drosophila. To further determine if hsp22 and hsp70 expression is correlated with lifespan, and if histone acetylation participates in this process, RNA levels for hsp22 and hsp70 were analyzed throughout the lifespan in the long-lived and short-lived iso-female lines. The results showed that hsp22 and hsp70 RNA levels were higher in long-lived line than in short-lived line and that the long-lived flies responded more rapidly to heat but were more tolerant to high temperature. Moreover, we investigated the influences of histone acetylation modification on longevity and on hsp gene expression by using histone deacetylase (HDAC) inhibitors TSA and BuA. The results demonstrated that both inhibitors were able to extend the lifespan and promote hsp22 and hsp70 expression. However, the optimal concentrations of these inhibitors, and probably the mechanisms of their actions, vary with the genetic background. In addition, we showed that HDAC inhibitors caused the hyperacetylation of core histone H3, implicating the involvement of chromatin modulation in hsp gene transcription. These data suggested a close correlation among histone acetylation, hsp gene expression and longevity in D. melanogaster.

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

NAD(P) has long been known as an essential energy-carrying molecule in cells. Recent data, however, indicate that NAD(P) also plays critical signaling roles in regulating cellular functions. The crystal structure of a human protein, HSCARG, with functions previously unknown, has been determined to 2.4-Å resolution. The structure reveals that HSCARG can form an asymmetrical dimer with one subunit occupied by one NADP molecule and the other empty. Restructuring of its NAD(P)-binding Rossmann fold upon NADP binding changes an extended loop to an α-helix to restore the integrity of the Rossmann fold. The previously unobserved restructuring suggests that HSCARG may assume a resting state when the level of NADP(H) is normal within the cell. When the NADP(H) level passes a threshold, an extensive restructuring of HSCARG would result in the activation of its regulatory functions. Immunofluorescent imaging shows that HSCARG redistributes from being associated with intermediate filaments in the resting state to being dispersed in the nucleus and the cytoplasm. The structural change of HSCARG upon NADP(H) binding could be a new regulatory mechanism that responds only to a significant change of NADP(H) levels. One of the functions regulated by HSCARG may be argininosuccinate synthetase that is involved in NO synthesis.

An NADPH Sensor Protein (HSCARG) Down-regulates Nitric Oxide Synthesis by Association with Argininosuccinate Synthetase and Is Essential for Epithelial Cell Viability

NADPH is an important cofactor in many biosynthesis pathways that control fundamental cellular processes. We recently determined the crystal structure of HSCARG, with functions previously unknown, and demonstrated it is an NADPH sensor, which undergoes restructuring and redistribution in response to changes of intracellular NADPH/NADP levels. In this study, we identified argininosuccinate synthetase (AS), a rate-limiting enzyme in nitric oxide synthesis, as capable of associating with HSCARG and demonstrated further that HSCARG decreased nitric oxide synthesis by down-regulating AS activity, whereas AS overexpression up-regulated hscarg mRNA transcription, suggesting a negative feedback mechanism. A decrease in the NADPH/NADP+ ratio, induced by dehydroepiandrosterone treatment, enhanced the interaction between HSCARG and AS, which resulted in stronger inhibition of AS activity and nitric oxide production. The dimerization region of HSCARG, amino acids 153-189, was identified to undergo critical interactions with AS. Furthermore, the viability of HSCARG RNA interference-treated epithelial cells decreased significantly, accompanied by an increase of the activity of caspase-3, which suggested that the loss of viability was because of apoptosis. These results indicate that HSCARG regulation of AS activity is crucial for maintaining the intracellular balance between redox state and nitric oxide levels.

Histone acetylation is involved in hsp70 gene transcription regulation in Drosophila melanogaster

The acetylation/deacetylation modifications of N-terminal tails of core histones play critical roles in activation/repression of many eukaryotic genes. However, the role of acetylation in transcription regulation of heat shock protein genes (hsp) is still a disputed issue. In this study, we investigated the influences of histone acetylation modification on changes in structure of polytene chromosomes and in expression of hsp70 gene in Drosophila melanogaster, by using histone deacetylase (HDAC) inhibitors Trichostatin A (TSA) and sodium butyrate (BuA), and the heat shock treatment of larvae of the flies. The results presented in this paper demonstrate that both TSA and BuA were able to affect the chromatin structure at the site where hsp70 gene is located along the polytene chromosome. Furthermore, the HDAC inhibitors significantly promoted the hsp70 gene transcription, at an extent similar to that induced by heat shock. The immunofluorescence in situ localization study further confirmed that the hsp70 gene locus was hyperacetylated after the heat induction. We therefore conclude that histone acetylation can significantly enhance both the basal and the inducible expression of hsp70 gene in D. melanogaster and hence plays important roles in hsp gene regulation. This study has provided a basis and a framework for further investigations aimed at the establishment of the correlation between acetylation modification and hsp gene regulation.

Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin)

Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated.

Characterisation of Caenorhabditis elegans sperm transcriptome and proteome.

BackgroundAlthough sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome.ResultsFirst, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway.ConclusionsTogether, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans.

Structure of Human Stabilin-1 Interacting Chitinase-like Protein (SI-CLP) Reveals a Saccharide-binding Cleft with Lower Sugar-binding Selectivity

Human secreted protein stabilin-1 interacting chitinase-like protein (SI-CLP) has been identified as a novel member of Glyco_18 domain-containing proteins that is involved in host defense and inflammatory reactions. Efficient secretion of SI-CLP is mediated by its interaction with the endocytic/sorting receptor stabilin-1. SI-CLP is expressed abundantly in macrophages and neutrophils and is up-regulated by Th2 cytokine IL-4 and glucocorticoid, which suggest that SI-CLP could be a marker for adverse effects of glucocorticoid therapy. To gain insight into the biological function of SI-CLP, we determined the crystal structure of SI-CLP at 2.7 Å resolution by x-ray crystallography and found that it featured a typical triose-phosphate isomerase barrel fold with a putative saccharide-binding cleft. Comparison with other chitinase-like proteins showed the cleft to be atypically wide and open. The saccharide-binding capacity of SI-CLP was investigated, and its ligand-binding specificity was found to relate to the length of the oligosaccharides, with preference for chitotetraose. Further investigations reveal that SI-CLP could bind LPS in vitro and neutralize its endotoxin effect on macrophages. Our results demonstrate the saccharide-binding property of SI-CLP by structure and in vitro biochemical analyses and suggest the possible roles of SI-CLP in pathogen sensing and endotoxin neutralization.

A novel nuclear‐localized protein with special adenylate kinase properties from Caenorhabditis elegans

The adrenal gland protein AD‐004 like protein (ADLP) from Caenorhabditis elegans was cloned and expressed in Escherichia coli. Enzyme assays showed that ADLP has special adenylate kinase (AK) properties, with ATP and dATP as the preferred phosphate donors. In contrast to all other AK isoforms, AMP and dAMP were the preferred substrates of ADLP; CMP, TMP and shikimate acid were also good substrates. Subcellular localization studies showed a predominant nuclear localization for this protein, which is different from AK1–AK5, but similar to that of human AK6. These results suggest that ADLP is more likely a member of the AK6 family. Furthermore, RNAi experiments targeting ADLP were conducted and showed that RNAi treatment resulted in the suppression of worm growth.

Transformation: how do nematode sperm become activated and crawl?

Nematode sperm undergo a drastic physiological change during spermiogenesis (sperm activation). Unlike mammalian flagellated sperm, nematode sperm are amoeboid cells and their motility is driven by the dynamics of a cytoskeleton composed of major sperm protein (MSP) rather than actin found in other crawling cells. This review focuses on sperm from Caenorhabditis elegans and Ascaris suum to address the roles of external and internal factors that trigger sperm activation and power sperm motility. Nematode sperm can be activated in vitro by several factors, including Pronase and ionophores, and in vivo through the TRY-5 and SPE-8 pathways. Moreover, protease and protease inhibitors are crucial regulators of sperm maturation. MSP-based sperm motility involves a coupled process of protrusion and retraction, both of which have been reconstituted in vitro. Sperm motility is mediated by phosphorylation signals, as illustrated by identification of several key components (MPOP, MFPs and MPAK) in Ascaris and the characterization of GSP-3/4 in C. elegans.

NADPH Is an Allosteric Regulator of HSCARG

NADP(H) is an important cofactor that controls many fundamental cellular processes. We have determined the crystal structure of HSCARG, a novel NADPH sensor, and found that it forms an asymmetrical dimer with only one subunit occupied by an NADPH molecule, and the two subunits have dramatically different conformations. To study the role of NADPH in affecting the structure and function of HSCARG, here, we constructed a series of HSCARG mutants to abolish NADPH binding ability. Protein structures of two mutants, R37A and Y81A, were solved by X-ray crystallography. The dimerization of wild-type and mutant HSCARG was studied by dynamic light scattering. Differences between the function of wild-type and mutant HSCARG were also compared. Our results show that binding of NADPH is necessary for HSCARG to form a stable asymmetric dimer. The conformation of the monomeric mutants was similar to that of NADPH-bound Molecule I in wild-type HSCARG, although some conformational changes were found in the NADPH binding site. Furthermore, we also noticed that abolition of NADPH binding ability changes the distribution of HSCARG in the cell and that these mutants without NADPH are more strongly associated with argininosuccinate synthetase as compared with wild-type HSCARG. These data suggest that NADPH functions as an allosteric regulator of the structure and function of HSCARG. In response to the changes in the NADPH/NADP(+) ratio within cells, HSCARG, as a redox sensor, associates and dissociates with NADPH to form a new dynamic equilibrium. This equilibrium, in turn, will tip the dimerization balance of the protein molecule and consequently controls the regulatory function of HSCARG.