Nuclear Import of Ho Endonuclease Utilizes Two Nuclear Localization Signals and Four Importins of the Ribosomal Import System *

Activity of Ho, the yeast mating switch endonuclease, is restricted to a narrow time window of the cell cycle. Ho is unstable and despite being a nuclear protein is exported to the cytoplasm for proteasomal degradation. We report here the molecular basis for the highly efficient nuclear import of Ho and the relation between its short half-life and passage through the nucleus. The Ho nuclear import machinery is functionally redundant, being based on two bipartite nuclear localization signals, recognized by four importins of the ribosomal import system. Ho degradation is regulated by the DNA damage response and Ho retained in the cytoplasm is stabilized, implying that Ho acquires its crucial degradation signals in the nucleus. Ho arose by domestication of a fungal VMA1 intein. A comparison of the primary sequences of Ho and fungal VMA1 inteins shows that the Ho nuclear localization signals are highly conserved in all Ho proteins, but are absent from VMA1 inteins. Thus adoption of a highly efficient import strategy occurred very early in the evolution of Ho. This may have been a crucial factor in establishment of homothallism in yeast, and a key event in the rise of the Saccharomyces sensu stricto. Ho endonuclease initiates a mating type switch in Saccharomyces cerevisiae and related yeasts by making a site-specific double strand break in a 24-bp cognate site in the mating type gene, MAT. Repair of the double strand break is by gene conversion using one of the silent cassettes of mating type information (HML␣ or HMRa) as a template. Repair occurs before replication of the MAT locus and each daughter cell has the new mating type with a regenerated Ho cognate site (1). Ho activity is tightly regulated: HO is transcribed briefly at the end of G 1 , its transcription is restricted to haploid mother cells, i.e. cells that have divided at least once (2), and the protein is rapidly degraded by the ubiquitin-26 S proteasome system (3). Cells in which Ho is retained in the nucleus beyond its normal time window of activity show perturbation of the cell cycle (4). Ho is marked for degradation by functions of the DNA damage response (DDR), 7 specifically the MEC1, RAD9, and CHK1 pathway (5). Despite being a nuclear protein, Ho must exit the nucleus to be degraded in the proteasomes. The DDR functions are important for Ho phosphorylation: phosphorylation of threonine 225 is crucial for Ho nuclear export and additional phosphorylations are required for recruitment of Ho for ubiquitylation. Ho is ubiquitylated by the SCF (Skp1-Cdc53-F-box protein) E3 ubiquitin ligase complex, to which it is recruited by the F-box protein Ufo1 (6). In mec1 mutants Ho is stabilized and accumulates in the nucleus; conversely trapping Ho in the nucleus by deletion of its nuclear exportin, Msn5, leads to stabilization of the protein (4). Ddi1 binds ubiquitylated Ho and is required for interaction of Ho with the proteasome; in its absence Ho is stabilized. The finding that Ho is not degraded within the nucleus, but in the cytoplasm, is further strengthened by the direct demonstration of accumulation of ubiquitylated Ho in the cytoplasm of ⌬ddi1 mutants (7). Ho nuclear import is very rapid and efficient. Ectopic expression of HO leads to rapid cleavage of MAT (8), and to a mating type switch at any phase of the cell cycle in both mother and daughter cells. This indicates that there is no impediment to its nuclear import (9). Macromolecules are conveyed through nuclear pore complexes in the nuclear envelope by soluble karyopherins. Karyopherins comprise two structurally related families, ␣-and ␤-karyopherins. These recognize specific nuclear localization sequence (NLS) peptide motifs in the cargo molecule: NLSs may comprise a short stretch of basic residues (classical/ cNLS), or two basic clusters 10 -12 residues apart (bipartite NLS) (10). Cargoes may be recognized by an adaptor protein, ␣-karyopherin/Srp1, which mediates their binding to the transport receptor, ␤-karyopherin/ Kap95 (11). Additionally, a family of about 14 ␤-karyopherins bind an array of cargoes directly and also makes contacts with the nucleoporin subunits of the nuclear pore complexes. Directionality of transport is determined by interaction with the GTPase Ran/yeast Gsp1. RanGTP is at a high concentration in the nucleus due to the asymmetric distribution of the Ran regulators. The nuclear guanine nucleotide exchange factor, RanGEF/yeast Prp20, converts RanGDP to RanGTP, whereas the GTPase activating protein, RanGAP/yeast Rna1, is localized in the cytoplasm and catalyzes the hydrolysis of RanGTP. Importin-cargo complexes assemble in the cytoplasm and after translocation into the nucleus they dissociate upon binding of RanGTP to the importin (12). To investigate how the efficient nuclear import that supports the unique biological function of Ho is achieved we located and analyzed its nuclea

PINCH in the cellular stress response to tau-hyperphosphorylation.

Particularly interesting new cysteine- histidine- rich protein (PINCH) is an adaptor protein that our data have shown is required for neurite extension under stressful conditions. Our previous studies also report that PINCH is recalled by neurons showing decreased levels of synaptodendritic signaling proteins such as MAP2 or synaptophysin in the brains of human immunodeficiency virus (HIV) patients. The current study addressed potential role(s) for PINCH in neurodegenerative diseases. Mass spectrometry predicted the interaction of PINCH with Tau and with members of the heat shock response. Our in vitro data confirmed that PINCH binds to hyperphosphorylated (hp) Tau and to E3 ubiquitin ligase, carboxy-terminus of heat shock-70 interacting protein. Silencing PINCH prior to induction of hp-Tau resulted in more efficient clearance of accumulating hp-Tau, suggesting that PINCH may play a role in stabilizing hp-Tau. Accumulation of hp-Tau is implicated in more than 20 neuropathological diseases including Alzheimer's disease (AD), frontotemporal dementia (FTD), and human immunodeficiency virus encephalitis (HIVE). Analyses of brain tissues from HIVE, AD and FTD patients showed that PINCH is increased and binds to hp-Tau. These studies address a new mechanism by which AD and HIV may intersect and identify PINCH as a contributing factor to the accumulation of hyperphosphorylated Tau

CCL8/MCP-2 is a target for mir-146a in HIV-1-infected human microglial cells

MicroRNA-mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir-146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir-146a, the chemokine CCL8/MCP-2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5-mediated HIV-1 entry and replication. In the present study, we have analyzed changes in the expression of mir-146a in primary human fetal microglial cells upon infection with HIV-1 and found increased expression of mir-146a. We further show that CCL8/MCP-2 is a target for mir-146a in HIV-1 infected microglia, as overexpression of mir-146a prevented HIV-induced secretion of MCP-2 chemokine. The clinical relevance of our findings was evaluated in HIV-encephalitis (HIVE) brain samples in which decreased levels of MCP-2 and increased levels of mir-146a were observed, suggesting a role for mir-146a in the maintenance of HIV-mediated chronic inflammation of the brain.—Rom, S., Rom, I., Passiatore, G., Pacifici, M., Radhakrishnan, S., Del Valle, L., Piña-Oviedo, S., Khalili, K., Eletto, D., Peruzzi, F. CCL8/MCP-2 is a target for mir-146a in HIV-1 infected human microglial cells

Activation of HIV-1 LTR by Rad51 in microglial cells

Infection with HIV-1 induces a variety of biological alterations to the host that are beneficial to the life cycle of the virus but may have adverse effects on the host cell. Here we demonstrate that expression of Rad51, a major component of the homologous recombination-directed DNA repair (HRR) pathway, is induced upon HIV-1 infection of microglial cells. Activation of Rad51 expression positively impacts on HIV-1 LTR transcription through a region of the viral promoter known for binding the inducible transcription factor NFκB. Rad51 showed the ability to form a complex with the p65 subunit of NFκB and regulate the level of p65 interaction with LTR DNA encompassing the κB motif. This study provides evidence for reciprocal interaction of HIV-1 and a host DNA repair protein that impacts on expression of the viral genome. These results also point to the ability of HIV-1 to recruit proteins involved in DNA repair that are necessary for retroviral DNA integration, efficient replication and prevention of viral-induced cell death

Expression levels of hp-Tau and PINCH in brain tissue from human Tau transgenic mouse.

<p>A) Western analyses of anterior frontal cortex (Ant-Fc), ventro-lateral posterior cortex (V-L-post-FC), posterior frontal cortex (post-FC), cerebellum (CB). B) Double immunofluorescence labeling of PINCH (green) and hp-Tau (red), co-localization (yellow) in hippocampal tissue from a wild-type mouse and the Tau-Tg mouse and respectively.</p

PINCH deletion mutants and sequences.

<p>PINCH deletion mutants and sequences.</p

PINCH levels in relation to changes in Tau solubility.

<p>The proteins from frontal cortex homogenates from normal, different Braak stages (1, 3, 5) of Alzheimer's disease (AD), HIV encephalitis (HIVE), and frontotemporal dementia (FTD) patients were fractionated into RAB (most soluble, RB), RIPA (less soluble, RB) and formic acid (FA) (least soluble). Representative Western blots from A) normal control, AD, HIVE, and FTD showing levels of PINCH, hpTau (s396) and total Tau (HT7). B) Coomassie stained gel of the same cases indicating total protein in each fraction. Compared to the control, increased levels of hp-Tau and PINCH are observed in disease cases. Loss of Tau and PINCH solubility are apparent in AD, HIVE and FTD, as well. Arrow in the FTD case indicates a blank lane in the gel.</p

Exposure of neurons to TNF-α results in increased levels of PINCH, hp-Tau and CHIP.

<p>Representative Western blot of A) neurons exposed to TNF-α; B) graphic representation of fold change of PINCH1, hp-Tau, total Tau and CHIP levels in TNF-α treated neurons over control. Results are from 4 separate experiments and are expressed as fold change over control. * p<0.005, **p<0.001 by one-way ANOVA with Tukey-Kramer post-hoc analyses. Grb-2 was used as the loading control. C) Fold change in hp-Tau/Total Tau * p = 0.0293 by one-tailed paired T-test.</p

Reciprocal immunoprecipitations indicate PINCH interacts with hp-Tau and CHIP.

<p>Neuronal lysate was immunoprecipitated with anti PINCH and reacted with antibodies against hp-Tau A) AT100 and B) AT8. C) Neuronal lysate was immunoprecipitated with anti-AT8 antibody and reacted with anti-PINCH, anti-Hsp90, anti-Hsp70 and CHIP. D) Neuronal lysate was immunoprecipitated with anti-PINCH antibody and reacted with anti-Hsp90, anti-Hsp70, anti-CHIP and anti-ILK. E) Reciprocal IP with anti-CHIP and reaction with anti-PINCH. F) Neuronal lysate was treated with (+) or without (−) phosphatase and immunoprecipitated with anti-HT7 antibody against total Tau and reacted with anti-PINCH antibody. G) The same neuronal lysate with (+) and without (−) phosphatase was immunoblotted with anti-HT7 and anti-PINCH antibodies without immunoprecipitation. Arrows indicate immunoreactive bands. Hc, heavy chain; lc, light chain; No IP (lysate), beads only (IgG); immunoprecipitation (IP).</p

Silencing PINCH during hp-Tau induction results in less hp-Tau accumulation.

<p>A) Representative Western blots of neurons uninfected (con), infected with shRNA against PINCH1 and 2 (shRNA P1/2) or non-target control shRNA with and without okadaic acid (OA) treatment. B) Quantification of protein expression levels of hp-Tau (AT8), and C) PINCH relative to loading control (GAPDH). No changes were detected in Hsp90, Hsp70 or CHIP. Results are from 4 separate experiments and are expressed as fold change over uninfected neurons (con). * p<0.005, **p<0.001 by one-way ANOVA with Tukey-Kramer post-hoc analyses.</p