Sex Differences in Mouse Hippocampal Astrocytes after <em>In-Vitro</em> Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

University of Wisconsin

Exposure of CV-1P cells to hypoxic conditions causes cell proliferation inhibition concomitant with the accumulation of pRb in the hypophosphorylated, growth suppressive form. This is in part due to inhibition of pRb-directed cdk4 and cdk2 activity. In this study we attempted to elucidate the mechanism by which cdk4 is inactivated under hypoxic conditions. After 18 h of hypoxia, CV-1P cells are inhibited from progressing from G(1) phase into the S phase of the cell cycle. This occurs in conjunction with dephosphorylation of serine-795, which is a putative substrate of cdk4. The amounts of cdk4, cdk6, and the D type cyclins are not affected by 18 h of hypoxia. The levels of cdki p16, p18, p19, and p57 under aerobic or hypoxic conditions were analyzed and although the levels of most cdki are unaffected by hypoxic conditions, the level of p16 increases significantly by 18 h of hypoxia. The mechanism by which cdk4 activity is inhibited under hypoxic conditions may be mediated through p16 association with cdk4. Immunoprecipitation analysis shows that p16 binds to cdk4 under hypoxic conditions but does not in cells maintained under aerobic conditions. Thus p16 may be involved in hypoxia-induced growth inhibition.

Hypoxia stimulates p16 expression and association with cdk4.

Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells. It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates. The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a. One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb). In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb. We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c. These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.

PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity.

The 63-kDa fragment of the protective antigen (PA) component of anthrax toxin forms a heptameric channel, (PA63)7, in acidic endosomal membranes that leads to the translocation of edema factor (EF) and lethal factor (LF) to the cytosol. It also forms a channel in planar phospholipid bilayer membranes. What role does this channel play in the translocation of EF and LF? We report that after the 263-residue N-terminal piece of LF (LFN) binds to its receptor on the (PA63)7 channel and its N-terminal end enters the channel at small positive voltages to block it, LFN is translocated through the channel to the opposite side at large positive voltages, thereby unblocking it. Thus, all of the translocation machinery is contained in the (PA63)7 channel, and translocation does not require any cellular proteins. The kinetics of this translocation are S-shaped, voltage-dependent, and occur on a timescale of seconds. We suggest that the translocation process might be explained simply by electrophoresis of unfolded LFN through the channel, but the refolding of the N-terminal half of LFN as it emerges from the channel may also provide energy for moving the rest of the molecule through the channel.

Protein translocation through anthrax toxin channels formed in planar lipid bilayers.

Phosphatase of regenerating liver 3 (PRL3) is overexpressed in a variety of tumors, and high levels of PRL3 expression are associated with tumorigenesis and metastasis. Consistent with an oncogenic role for PRL3, we show that ectopic PRL3 expression promotes cell proliferation and invasion. However, little is known about the molecular basis for PRL3 function. Obtaining this knowledge is vital for understanding PRL3-mediated disease processes and for the development of novel anticancer therapies targeted to PRL3. Here we report that up-regulation of PRL3 activates the Src kinase, which initiates a number of signal pathways culminating in the phosphorylation of ERK1/2, STAT3, and p130(Cas). The activation of these pathways likely contributes to the increased cell growth and motility of PRL3 cells. We provide evidence that PRL3 induces Src activation through down-regulation of Csk, a negative regulator of Src. Importantly, Src activation and Csk down-regulation are also observed in colon cancer cells expressing a higher level of PRL3. Thus, we have revealed a biochemical mechanism for the PRL3-mediated cell invasion and proliferation in which elevated PRL3 expression causes a reduction in Csk level, leading to Src activation.

PRL3 promotes cell invasion and proliferation by down-regulation of Csk leading to Src activation.

Micronutrients such as siderophore-bound iron and vitamin B(12) cross the outer membrane of gram-negative bacteria through a group of 22-stranded beta-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the beta-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes.

Intimins and invasins are virulence factors produced by pathogenic Gram-negative bacteria. They contain C-terminal extracellular passenger domains that are involved in adhesion to host cells and N-terminal Î² domains that are embedded in the outer membrane. Here, we identify the domain boundaries of an E.Â coli intimin Î² domain and use this information to solve its structure and the Î² domain structure ofÂ a Y.Â pseudotuberculosis invasin. Both Î² domain structures crystallized as monomers and reveal that the previous range of residues assigned to the Î² domain also includes a protease-resistant domain that is part of the passenger. Additionally, we identify 146 nonredundant representative members of the intimin/invasin family based on the boundaries of the highly conserved intimin and invasin Î² domains. We then use this set of sequences along with our structural data to find and map the evolutionarily constrained residues within the Î² domain.

Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis.

Intimins et invasins sont des facteurs de virulence produites par des bact&eacute;ries pathog&egrave;nes &agrave; Gram n&eacute;gatif. Ils contiennent des domaines de passager extracellulaire C-terminaux qui sont impliqu&eacute;es dans l&#39;adh&eacute;rence aux cellules h&ocirc;tes et domaines &beta;-N-terminale qui sont incorpor&eacute;es dans la membrane externe. Ici, nous identifions les limites de domaine d&#39;un domaine &beta; intimine e. coli et utiliser ces informations pour r&eacute;soudre sa structure et la structure de domaine &beta; d&#39;un Y. pseudotuberculosis invasin. Les deux structures de domaine &beta; cristallise sous forme de monom&egrave;res et r&eacute;v&egrave;lent que la pr&eacute;c&eacute;dente gamme des r&eacute;sidus assign&eacute;s au domaine &beta; comprend &eacute;galement un domaine prot&eacute;ase-r&eacute;sistant qui fait partie du passager. En outre, nous identifions 146 membres repr&eacute;sentatifs non redondante de la famille intimine/invasin bas&eacute; sur les limites de l&#39;intimine hautement conserv&eacute;e et domaines &beta; invasin. Nous utilisons ensuite cet ensemble de s&eacute;quences avec nos donn&eacute;es structurales de trouver et de cartographier les r&eacute;sidus &eacute;volutivement contraints au sein du domaine &beta;.

Structures cristallines du domaine la membrane externe de l&#39;intimine et invasin d&#39;Escherichia coli ent&eacute;ro-h&eacute;morragique et ent&eacute;ropathog&egrave;nes Y. pseudotuberculosis.

Intimins e invasins sono fattori di virulenza, prodotti da batteri patogeni gram-negativi. Essi contengono domini extracellulari passeggero C-terminale che sono coinvolti nell&#39;adesione alle cellule ospiti e domini &beta;-N-terminale che sono incorporati nella membrana esterna. Qui, ci identifichiamo i confini del dominio di un dominio di e. coli intimin &beta; e utilizzare queste informazioni per risolvere la sua struttura e la struttura di dominio &beta; di una Y. pseudotuberculosis invasin. Entrambe le strutture &beta; dominio cristallizzato come monomeri e rivelano che la precedente gamma di residui assegnato al dominio &beta; include anche un dominio resistente alle proteasi che &egrave; parte del passeggero. Inoltre, ci identifichiamo 146 nonredundant membri rappresentativi della famiglia intimin/invasin basato sui confini del intimin altamente conservata e domini &beta; invasin. Quindi usiamo questo set di sequenze insieme ai nostri dati strutturali per individuare e mappare i residui evolutivamente vincolati all&#39;interno del dominio &beta;.

Strutture di cristallo del dominio membrana esterna di intimin e invasin da e. coli enteroemorragica ed enteropatogeno Y. pseudotuberculosis.

TonB-dependent transporters (TBDTs), which transport iron-chelating siderophores and vitamin B(12) across the outer membrane of Gram-negative bacteria, share a conserved architecture of a 22-stranded β-barrel with an amino-terminal plug domain occluding the barrel. We previously reported that we could induce TBDTs to reversibly open in planar lipid bilayers via the use of urea and that these channels were responsive to physiological concentrations of ligands. Here we report that in the presence of urea, trypsin can cleave the amino-terminal 67 residues of the plug of the TonB-dependent transporter FhuA, as assessed by gel shift and mass spectrometry assays. On the bilayer, trypsin treatment in the presence of urea resulted in the induced conductance no longer being reversed upon removal of urea, suggesting that urea opens intact FhuA channels by pulling the plug at least partly out of the barrel and that removal of the urea then allows reinsertion of the plug into the barrel. When expressed separately, the FhuA plug domain was found to be a mostly unfolded structure that was able to occlude isolated FhuA β-barrels inserted into the membrane. Thus, although folded in the barrel, the plug need not be folded upon exiting the barrel. The rate of insertion of the β-barrels into the membrane was tremendously increased in the presence of an osmotic gradient provided by either urea or glycerol. Negative staining electron microscopy showed that FhuA in a detergent solution formed vesicles, thus explaining why an osmotic gradient promoted the insertion of FhuA into membranes.

TonB-dependent transporter FhuA in planar lipid bilayers: partial exit of its plug from the barrel.

Male neonate brains are more susceptible to the effects of perinatal asphyxia resulting in hypoxia and ischemia (HI)-related brain injury. The relative resistance of female neonatal brains to adverse consequences of HI suggests that there are sex-specific mechanisms that afford females greater neuroprotection and/or facilitates recovery post-HI. We hypothesized that HI preferentially induces estrogen receptor α (ERα) expression in female neonatal hippocampi and that ERα is coupled to Src family kinase (SFK) activation that in turn augments phosphorylation of the TrkB and thereby results in decreased apoptosis. After inducing the Vannucci's HI model on P9 (C57BL/6J) mice, female and male ERα wild-type (ERα(+/+)) or ERα null mutant (ERα(-/-)) mice received vehicle control or the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF). Hippocampi were collected for analysis of mRNA of ERα and BDNF, protein levels of ERα, p-TrkB, p-src, and cleaved caspase 3 (c-caspase-3) post-HI. Our results demonstrate that: (1) HI differentially induces ERα expression in the hippocampus of the female versus male neonate, (2) src and TrkB phosphorylation post-HI is greater in females than in males after 7,8-DHF therapy, (3) src and TrkB phosphorylation post-HI depend on the presence of ERα, and (4) TrkB agonist therapy decreases the c-caspase-3 only in ERα(+/+) female mice hippocampus. Together, these observations provide evidence that female-specific induction of ERα expression confers neuroprotection with TrkB agonist therapy via SFK activation and account for improved functional outcomes in female neonates post-HI.

Eshwar Udho

Waisman Center

University of Wisconsin

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Eshwar Udho

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University of Wisconsin

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Waisman Center