My-Nuong Vo

Retroviral Nucleocapsid Proteins Display Nonequivalent Levels of Nucleic Acid Chaperone Activity

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NCs chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NCs highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 ∼ RSV > MLV ≫ HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single- and double-stranded oligonucleotides suggested that all four NC proteins have moderate duplex destabilization capabilities. Single-molecule DNA-stretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.

Differential effects of phthalates on the testis and the liver.

Abstract Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) α is equally abundant in the liver and the testis, whereas PPARγ and retinoic acid receptor (RAR) α are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RARα and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPARα and PPARγ in Sertoli cells, but they decreased the nuclear localization of RARα, as previously shown. Both PPARα and PPARγ were in the nucleus and cytoplasm of liver cells, but RARα was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RARα, PPARα, and PPARγ in these organs.

Ligand-Dependent Regulation of Retinoic Acid Receptor α in Rat Testis: In Vivo Response to Depletion and Repletion of Vitamin A

Male animals are sterile due to testicular degeneration in the absence of retinoic acid (RA) or functional retinoic acid receptor-alpha (RAR alpha). This degeneration can be reversed by injecting retinol, a precursor of RA, into vitamin A-deficient (VAD) rats. To determine the relationship between this ligand-dependent testicular degeneration and regeneration and the expression levels of RAR alpha messenger RNA and protein, testes were depleted and then replenished with retinol in vivo. Results showed that RAR alpha messenger RNA and protein levels declined to VAD amounts after 7 weeks on a VAD diet. This decline was due to decreased RAR alpha levels in early meiotic spermatocytes and the loss of advanced germ cells. Interestingly, the advanced germ cells still contained RAR alpha, but the protein was primarily cytoplasmic instead of nuclear, indicating inactivity as a transcription factor. In VAD testis, RAR alpha levels were low and then increased primarily in Sertoli cells after retinol replenishment. TUNEL analyses showed that most germ cells at the basal aspect of seminiferous tubules were undergoing apoptosis during degeneration. These results indicate that RAR alpha is either down-regulated or inactivated in RA-deficient testis and coincident with that, testes degenerate by apoptosis or selective loss of germ cells.

Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are “chaperoned” by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a “Y” shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the “Y” conformation.

Deficiencies in tRNA synthetase editing activity cause cardioproteinopathy.

Significance Misfolded proteins are a hallmark of diverse human cardiac disorders including desmin-related cardiomyopathy and systemic amyloidosis. Defects in translational fidelity can cause neurodegeneration, however the consequences of mistranslation in other tissues, including the heart, are unknown. The fidelity of protein synthesis is largely ensured by aminoacyl-tRNA synthetases, and many tRNA synthetases contain editing domains that hydrolyze mischarged tRNAs, preventing incorporation of incorrect amino acids into proteins. Here, we show that diminished editing efficacy of the alanyl-tRNA synthetase causes misfolded protein aggregation and cell death in the mammalian heart. These results illuminate the importance of translational fidelity in cardiac homeostasis and suggest that genetic factors that disrupt the accuracy of translation may contribute to proteinopathies of the heart and other tissues. Misfolded proteins are an emerging hallmark of cardiac diseases. Although some misfolded proteins, such as desmin, are associated with mutations in the genes encoding these disease-associated proteins, little is known regarding more general mechanisms that contribute to the generation of misfolded proteins in the heart. Reduced translational fidelity, caused by a hypomorphic mutation in the editing domain of alanyl-tRNA synthetase (AlaRS), resulted in accumulation of misfolded proteins in specific mouse neurons. By further genetic modulation of the editing activity of AlaRS, we generated mouse models with broader phenotypes, the severity of which was directly related to the degree of compromised editing. Severe disruption of the editing activity of AlaRS caused embryonic lethality, whereas an intermediate reduction in AlaRS editing efficacy resulted in ubiquitinated protein aggregates and mitochondrial defects in cardiomyocytes that were accompanied by progressive cardiac fibrosis and dysfunction. In addition, autophagic vacuoles accumulated in mutant cardiomyocytes, suggesting that autophagy is insufficient to eliminate misfolded proteins. These findings demonstrate that the pathological consequences of diminished tRNA synthetase editing activity, and thus translational infidelity, are dependent on the cell type and the extent of editing disruption, and provide a previously unidentified mechanism underlying cardiac proteinopathy.

Effect of Mg2+ and Na+ on the Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein : Implications for Reverse Transcription

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is an essential protein for retroviral replication. Among its numerous functions, NC is a nucleic acid (NA) chaperone protein that catalyzes NA rearrangements leading to the formation of thermodynamically more stable conformations. In vitro, NC chaperone activity is typically assayed under conditions of low or no Mg(2+), even though reverse transcription requires the presence of divalent cations. Here, the chaperone activity of HIV-1 NC was studied as a function of varying Na(+) and Mg(2+) concentrations by investigating the annealing of complementary DNA and RNA hairpins derived from the trans-activation response domain of the HIV genome. This reaction mimics the annealing step of the minus-strand transfer process in reverse transcription. Gel-shift annealing and sedimentation assays were used to monitor the annealing kinetics and aggregation activity of NC, respectively. In the absence of protein, a limited ability of Na(+) and Mg(2+) cations to facilitate hairpin annealing was observed, whereas NC stimulated the annealing 10(3)- to 10(5)-fold. The major effect of either NC or the cations is on the rate of bimolecular association of the hairpins. This effect is especially strong under conditions wherein NC induces NA aggregation. Titration with NC and NC/Mg(2+) competition studies showed that the annealing kinetics depends only on the level of NA saturation with NC. NC competes with Mg(2+) or Na(+) for sequence-nonspecific NA binding similar to a simple trivalent cation. Upon saturation, NC induces attraction between NA molecules corresponding to approximately 0.3 kcal/mol/nucleotide, in agreement with an electrostatic mechanism of NC-induced NA aggregation. These data provide insights into the variable effects of NCs chaperone activity observed during in vitro studies of divalent metal-dependent reverse transcription reactions and suggest the feasibility of NC-facilitated proviral DNA synthesis within the mature capsid core.

Dissociating Quaternary Structure Regulates Cell-signaling Functions of a Secreted Human tRNA Synthetase

Many tRNA synthetases are homodimers that are catalytically inactive as monomers. An example is the 528-amino acid human tyrosyl-tRNA synthetase, which is made up of an N-terminal catalytic unit (TyrRSMini) and a 164-amino acid C-domain. Although native TyrRS has no known cytokine functions, natural proteolysis of secreted TyrRS releases TyrRSMini, which not only has the same aminoacylation activity as native TyrRS but also has strong activity for stimulating migration of polymorphonuclear leukocytes. The migration-stimulating activity is dependent on an ELR tripeptide motif, similar to that in CXC cytokines like IL-8, and also has the familiar bell-shaped concentration dependence seen for CXC cytokines. Here we show that in contrast to IL-8, where the bell-shaped dependence arises from the effects of CXCR1/2 receptor internalization, TyrRSMini does not induce internalization of CXCR1/2. A rationally designed non-associating monomer and a non-dissociating dimer were constructed. With these constructs, the bell-shaped concentration dependence of leukocyte migration was shown to arise from the agonist (for migration) activity of the catalytically inactive monomer and the antagonist activity of the catalytically active dimer. Thus, the dissociating quaternary structure of TyrRSMini regulates two opposing cytokine activities and suggests the possibility of dissociating quaternary structures regulating novel functions of other tRNA synthetases.

Peroxisome Proliferators Disrupt Retinoic Acid Receptor Alpha Signaling in the Testis

Abstract Peroxisome proliferators include a diverse group of chemicals, some of which have been demonstrated to be testicular toxicants. However, the mechanism by which peroxisome proliferators, such as phthalates, cause testicular damage is not clear. It is known that retinoic acid receptor alpha (RARα) and its retinoic acid ligand, the acid form of vitamin A, are required for spermatogenesis. It has been demonstrated that the absence of RARα gene or vitamin A in the animal leads to testis degeneration and sterility. Therefore, any compound that disrupts the action of vitamin A in the testis could potentially be damaging to male fertility. The current investigation examined a novel hypothesis that a mechanism of degeneration by peroxisome proliferators in the testis is due, in part, to disruption of the critical RARα signaling pathway. We show that peroxisome proliferators were able to disrupt the retinoic acid-induced nuclear localization of RARα and the retinoic acid-stimulated increase in transcriptional activity of a retinoic acid-responsive reporter gene in Sertoli cells. Concomitantly, peroxisome proliferators increased the nuclear localization of PPARα and the transcriptional activity of a peroxisome proliferator-responsive reporter gene in these cells. These results indicate that peroxisome proliferators can indeed shift the balance of nuclear localization for RARα and PPARα, resulting in deactivation of the critical RARα transcriptional activity in Sertoli cells.

Positive Regulation of Retinoic Acid Receptor Alpha by Protein Kinase C and Mitogen-Activated Protein Kinase in Sertoli Cells

Abstract Retinoic acid receptor α (RARα) is required for normal testis function. Similar to other steroid hormone receptors, RARα appears to undergo an activation process by which it translocates from the cytoplasm to the nucleus where it acts as a transcription factor. In this report, we demonstrate that RARα nuclear trafficking in Sertoli cells is positively regulated by phorbol-12-myristate-13-acetate-activated protein kinase C without the requirement of ligand, retinoic acid. Protein kinase C then stimulates the downstream mitogen-activated protein kinase, and the nuclear localization of RARα is dependent on activation of both kinases. The increase in RARα nuclear translocation is also coupled with enhanced transcriptional activity of RARα. This mechanism of RARα positive regulation is unique, different from that of its negative regulation, that has previously been shown to be dependent on cAMP-dependent protein kinase A and more importantly, dependent on its ligand. However, the mechanism by which retinoic acid positively influences the nuclear localization of RARα is not due to retinoic acid directly increasing protein kinase C or mitogen-activated protein kinase activities. Nonetheless, the positive influence of retinoic acid is also dependent on these two kinases as determined by inhibitor studies. These results suggest two mechanisms for RARα activation in Sertoli cells: one involving only the two kinases, the other involving both the ligand and the two kinases. These regulatory mechanisms for RARα activation, both positive and negative, may be critical for the proper function of RARα in the testis.

The N-terminal zinc finger and flanking basic domains represent the minimal region of the human immunodeficiency virus type-1 nucleocapsid protein for targeting chaperone function

The human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein is a chaperone that facilitates nucleic acid conformational changes to produce the most thermodynamically stable arrangement. The critical role of NC in many steps of the viral life cycle makes it an attractive therapeutic target. The chaperone activity of NC depends on its nucleic acid aggregating ability, duplex destabilizing activity, and rapid on-off binding kinetics. During the minus-strand transfer step of reverse transcription, NC chaperones the annealing of highly structured transactivation response region (TAR) RNA to the complementary TAR DNA. In this work, the role of different functional domains of NC in facilitating 59-nucleotide TAR RNA-DNA annealing was probed by using chemically synthesized peptides derived from full-length (55 amino acids) HIV-1 NC: NC(1-14), NC(15-35), NC(1-28), NC(1-35), NC(29-55), NC(36-55), and NC(11-55). Most of these peptides displayed significantly reduced annealing kinetics, even when present at concentrations much higher than that of wild-type (WT) NC. In addition, these truncated NC constructs generally bind more weakly to single-stranded DNA and are less effective nucleic acid aggregating agents than full-length NC, consistent with the loss of both electrostatic and hydrophobic contacts. However, NC(1-35) displayed annealing kinetics, nucleic acid binding, and aggregation activity that were very similar to those of WT NC. Thus, we conclude that the N-terminal zinc finger, flanked by the N-terminus and linker domains, represents the minimal sequence that is necessary and sufficient for chaperone function in vitro. In addition, covalent continuity of the 35 N-terminal amino acids of NC is critical for full activity. Thus, although the hydrophobic pocket formed by residues proximal to the C-terminal zinc finger has been a major focus of recent anti-NC therapeutic strategies, NC(1-35) represents an alternative target for therapeutics aimed at disrupting NCs chaperone function.