Sudden death due to paralysis and synaptic and behavioral deficits when <Emphasis Type="Italic">Hip14/Zdhhc17</Emphasis> is deleted in adult mice

Background

Palmitoylation, the addition of palmitate to proteins by palmitoyl acyltransferases (PATs), is an important regulator of synaptic protein localization and function. Many palmitoylated proteins and PATs have been implicated in neuropsychiatric diseases, including Huntington disease, schizophrenia, amyotrophic lateral sclerosis, Alzheimer disease, and X-linked intellectual disability. HIP14/DHHC17 is the most conserved PAT that palmitoylates many synaptic proteins. Hip14 hypomorphic mice have behavioral and synaptic deficits. However, the phenotype is developmental; thus, a model of post-developmental loss of Hip14 was generated to examine the role of HIP14 in synaptic function in the adult.

Results

Ten weeks after Hip14 deletion (iHip14 ^Δ/Δ ), mice die suddenly from rapidly progressive paralysis. Prior to death the mice exhibit motor deficits, increased escape response during tests of anxiety, anhedonia, a symptom indicative of depressive-like behavior, and striatal synaptic deficits, including reduced probability of transmitter release and increased amplitude but decreased frequency of spontaneous post-synaptic currents. The mice also have increased brain weight due to microgliosis and astrogliosis in the cortex.

Conclusions

Behavioral changes and electrophysiological measures suggest striatal dysfunction in iHip14 ^Δ/Δ mice, and increased cortical volume due to astrogliosis and microgliosis suggests a novel role for HIP14 in glia. These data suggest that HIP14 is essential for maintenance of life and neuronal integrity in the adult mouse.

     

Design,synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan’s poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC₅₀ value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.     

Mitochondrial apoptosis induced by BH3‐only molecules in the exclusive presence of endoplasmic reticular Bak

Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3‐only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3‐only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER‐stress surveillance machinery IRE1α/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3‐only mediators can rely on reticular Bak to activate an ER‐to‐mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax‐dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation.     

The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein

Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|{"type":"entrez-protein","attrs":{"text":"CCA61804.1","term_id":"379991184","term_text":"CCA61804.1"}}CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|{"type":"entrez-protein","attrs":{"text":"ADZ24001.1","term_id":"325513923","term_text":"ADZ24001.1"}}ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent K_d of 1.14 × 10⁻⁶ m⁻¹. We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes.     

The Escherichia coli Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage

The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.The Escherichia coli Dcm protein methylates the second C of CCWGG sites (W = A or T). Deamination of 5-methylcytosine converts CG base pairs to T/G mismatches, causing CCWGG-to-CTWGG transition mutations. Very-short-patch (VSP) repair minimizes these mutations (2). Repair is initiated by a sequence- and mismatch-specific endonuclease, Vsr, which cleaves the DNA 5′ of the T. DNA polymerase I removes the T along with a few 3′ nucleotides and resynthesizes the missing bases, restoring the CG base pair. Vsr is both necessary and sufficient for initiating VSP repair. However, two other proteins, MutS and MutL, enhance VSP repair of deamination damage (1).MutS and MutL are best known for their roles in postreplication mismatch repair (MMR) (9, 11). MutL couples mismatch recognition by MutS to the activation of MutH, an endonuclease that cleaves the unmethylated strand of GATC sequences that are transiently hemimethylated following DNA replication. The nicked strand, containing the erroneous base, is removed by the UvrD helicase and one of several exonucleases to beyond the mismatch and then resynthesized by DNA polymerase III.MutL stimulates the endonuclease activities of both Vsr and MutH in vitro (8, 17). The requirements for stimulation are the same: a mismatch, MutS, and ATP hydrolysis by MutL (8, 8a). Cross-linking studies showed that MutH and Vsr interact with the same region in the N-terminal domain of MutL (Heinze et al., submitted). Competition of Vsr with MutH for access to MutL explains the ability of Vsr to inactivate MMR in vivo when overexpressed (6, 13). Thus, the interactions of the two repair endonucleases with MutL are structurally and functionally very similar.In contrast to MMR, where the cleavage site for MutH may be several kilobases away from the mismatch, VSP repair requires that mismatch recognition and endonucleolytic cleavage occur at the same C(T/G)WGG site. How MutS and MutL stimulate VSP repair if MutS and Vsr compete for the same mismatch remains unknown (2, 12). We hypothesized that MutS binds the mismatch first and that a MutS-MutL complex then recruits Vsr. If so, then the MMR proteins would initially mask the mismatch, making the interaction of Vsr with MutL independent of lesion identity.To test this hypothesis, we studied the interaction of MutL with Vsr and with MutH in response to two types of mismatch by using a bacterial two-hybrid assay (10). This assay detects all known interactions among the Mut proteins: homodimerization of MutS and MutL, interaction of MutL with MutS and with MutH, and interaction of Vsr with the N-terminal domain of MutL (15). We found no false positives or false negatives. Furthermore, since the assay relies on reconstitution of a soluble protein (adenylate cyclase), the DNA repair proteins are free to interact with the DNA (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Known interactions among repair proteins as detected by the bacterial two-hybrid assay. The T18 and T25 subunits of CyaA are fused to any two repair proteins (illustrated here by MutL and Vsr), allowing measurement of all pairwise interactions as units of β-galactosidase (β-gal). T25 fusions are repair proficient. CRP, cyclic AMP (cAMP) receptor protein; P, lac operon promoter; RNAP, RNA polymerase.2-Aminopurine (2AP) mispairs with C during DNA replication, causing transition and frameshift mutations (5). The transitions are due primarily to the mismatch itself; the frameshifts are due to saturation of MMR, which leaves slipped-strand intermediates caused by DNA replication errors unrepaired (19). MutS and MutL bind to 2AP/C lesions (22), although the lesions may not be subject to MMR (19). As shown in Fig. ​Fig.2,2, treatment with 2AP causes a dose-dependent increase in the interaction of MutL with both Vsr and MutH; dimerization of MutL and interaction of MutL with MutS are somewhat increased.Open in a separate windowFIG. 2.Effect of 2AP treatment on protein-protein interactions in the bacterial two-hybrid assay. Results in units of β-galactosidase ± standard errors of the means (n = 9) are shown for BTH101(F galE15 ga1K16 rpsL1 hsdR2 mcrA1 mcrB1 cyaA-99) cells treated with 2AP as described previously (5, 19). Cells were cotransformed with pT18 and pT25 vectors (light gray bars), pT18-mutS and pT25-mutL (white bars), pT18-vsr and pT25-mutL (gray bars), pT18-mutH and pT25-mutL (black bars), or pT18-mutL and pT25-mutL (mottled bars). (NB: The dose-response curve for the pT18-mutS pT25-mutS transformants is similar to that of the pT18-mutL pT25-mutL transformants; it has been omitted for graphical clarity since the MutS-MutS interaction gives very high units of β-galactosidase activity [15]).The MutY adenine glycosylase removes A''s which have mispaired with oxidized guanine (8-oxoG) during DNA replication. Cells with a deletion of mutY have an elevated frequency of CG-to-AT transversion mutations (18); these are reduced by excess MutS, suggesting that 8-oxoG/A mismatches are also subject to MMR (23). As shown in Fig. ​Fig.3,3, the interactions between Vsr and MutL and between MutH and MutL increase in a mutY cell (stippled bars). Other interactions, such as MutS dimerization, are unaffected (not shown).Open in a separate windowFIG. 3.Effects of mutY and mutT deletions on protein-protein interactions in the bacterial two-hybrid assay. Results are in units of β-galactosidase, relative to the level in the wild type, in mutT (solid) and mutY (stippled) derivatives of BTH101 cotransformed with pT18 and pT25 vectors, pT18-mutH and pT25-mutL, pT18-vsr and pT25-mutL, or pT18-mutS and pT25-mutS (n = 3).8-OxoG/A mismatches also arise by incorporation of oxidized dGTP opposite A during DNA replication. The MutT nuclease minimizes this by removing oxidized dGTP from the nucleotide pool. The high frequency of AT-to-CG mutations in mutT strains is unaffected by the status of the MMR system (7, 21, 23), possibly because these 8-oxoG/A mispairs are in a conformation that MutS does not recognize. As shown in Fig. ​Fig.3,3, neither the interaction between MutL and Vsr nor that between MutL and MutH is elevated in a mutT strain (solid bars).These data show that mismatches which attract MutS and MutL increase the interaction of MutL with MutH in vivo. Although these mismatches are not subject to VSP repair, they also increase the interaction between MutL and Vsr. The simplest interpretation is that a MutS-MutL complex recruits MutH and Vsr to the DNA independent of the identity of the mismatch. MutS and MutL could then clear the mismatch, delivering the (activated) endonuclease to its specific target site, no matter how far away it is.Interaction of MutL with MutH, leading to MMR, is probably the default option. However, the MutS-MutL complex may recruit other repair proteins, such as Vsr or UvrB (20), to lesions that are poorly processed by MMR. The T/G mismatch in hemimethylated CTWGG sequences may be one such site. Vsr is expressed at very low levels in growing cells (14), so this recruitment would enhance VSP repair. However, recruitment of Vsr to other lesions would reduce VSP repair. For example, recruitment of Vsr by MutL to 2AP/C lesions (Fig. ​(Fig.2)2) could explain why CCWGG sites are hotspots for 2AP-induced mutations (4, 19).We have argued that Vsr is kept at low levels while DNA is replicating to avoid interference with MMR (14). However, if, as we suggest here, MutS and MutL are needed to recruit scarce Vsr to its target sequence, this argument loses its merit. It seems more likely that Vsr levels are kept low to avoid CTWGG-to-CCWGG mutations; Vsr creates these mutations by converting T/G mismatches formed at CTAGG sites by errors in DNA replication to CG (3, 6, 16). Vsr levels rise in nongrowing cells (14), when mutagenesis is no longer a risk. Under these circumstances, it is likely that MutS and MutL are no longer required for efficient VSP repair.     

PTEN Loss Promotes Mitochondrially Dependent Type II Fas-Induced Apoptosis via PEA-15

Two distinct biochemical signals are delivered by the CD95/Fas death receptor. The molecular basis for the differential mitochondrially independent (type I) and mitochondrially dependent (type II) Fas apoptosis pathways is unknown. By analyzing 24 Fas-sensitive tumor lines, we now demonstrate that expression/activity of the PTEN tumor suppressor strongly correlates with the distinct Fas signals. PTEN loss-of-function and gain-of-function studies demonstrate the ability to interconvert between type I and type II Fas pathways. Importantly, from analyses of Bcl-2 transgenic Pten^+/− mice, Pten haploinsufficiency converts Fas-induced apoptosis from a Bcl-2-independent to a Bcl-2-sensitive response in primary thymocytes and activated T lymphocytes. We further show that PTEN influences Fas signaling, at least in part, by regulating PEA-15 phosphorylation and activity that, in turn, regulate the ability of Bcl-2 to suppress Fas-induced apoptosis. Thus, PTEN is a key molecular rheostat that determines whether a cell dies by a mitochondrially independent type I versus a mitochondrially dependent type II apoptotic pathway upon Fas stimulation.Two types of Fas apoptotic signaling pathways, designated the type I and type II pathways, occur in distinct classes of cells (2). Biochemically, type I and type II cells differ primarily in the amounts of FADD and caspase-8 recruited to the Fas receptor, in the kinetics of caspase cascade activation, and in their relative dependence on the mitochondrial intrinsic arm of the Fas apoptotic pathway in the execution of cell death (34). Fas receptor aggregation leads to the recruitment of the adaptor protein FADD and the initiator caspase-8 and -10, forming the death-inducing signaling complex (DISC) and resulting in autoproteolytic activation of these caspases. In type I cells, a sufficient amount of caspase-8 is processed to directly activate the effector caspase-3 and to execute apoptosis. While the intrinsic mitochondrial apoptotic pathway is also activated in type I cells, the relative contribution of this branch to apoptosis induction is diminished by the potent action of the direct pathway. In contrast to type I cells and despite similar expression of cell surface Fas, type II cells form a weak DISC and exhibit delayed kinetics of caspase-8 and -3 activation. Due to the paucity of FADD recruitment and caspase-8 processing at the DISC in type II cells, the direct activation of caspase-3 is attenuated, resulting in the increased dependence of type II cells on the mitochondrial amplification loop activated by the proapoptotic Bcl-2 member Bid in order to execute apoptosis. Hence, type I cells undergo Fas-mediated apoptosis in a mitochondrially independent manner, whereas type II cells have increased dependence on the intrinsic mitochondrial pathway to induce apoptosis.Despite an intensive search, the identity of the signaling protein(s) that determines whether a cell dies by type I versus type II Fas-induced apoptosis has remained elusive (28). By virtue of their ability to regulate Fas signaling in various tissue types, a plethora of signaling proteins, including death receptor signaling proteins such as DAXX, FAP-1, FAF1, FLASH, RIP, and FLIP, apoptosis regulatory proteins such as IAP family members, Bcl-2-related proteins, and signaling proteins such as PP2A, CaMKII, PEA-15, galectin-3, PTEN, PI3K, and PKB, among others, have been implicated as potential candidates (8-11, 13-16, 21, 28, 42, 46).In search of the signaling pathway(s) that is differentially activated in type I and type II cells, we performed a Kinetworks phosphosite screen (KPSS1.3), which simultaneously detects the presence and relative quantities of 34 critical protein phosphorylation sites, and found that the serine/threonine protein kinase B (PKB; also known as Akt) was highly phosphorylated in prototypic type II Jurkat but not type I H9 cells (Kinexus, Vancouver, BC) (data not shown). Furthermore, we noted that both of the prototypic type II cell lines, i.e., Jurkat and CEM, are known to be deficient in the PTEN tumor suppressor (33). Therefore, we hypothesized that PTEN may be an important regulator of the differential Fas signaling pathways in type I and type II cells.The PTEN tumor suppressor gene is among the most commonly mutated genes in a broad range of human malignancies. PTEN is an important negative regulator of cell growth and survival. Among other functions, PTEN is a phosphatidylinositol 3′-phosphatase that specifically downmodulates the levels of phosphoinositide second messengers such as phosphatidylinositol(3,4,5)-trisphosphate, thereby antagonizing the action of phosphatidylinositol 3-kinase (PI3K). Loss of PTEN function results in increased membrane phosphatidylinositol(3,4,5)-trisphosphate levels and constitutive activation of its downstream effectors, such as PKB, leading to enhanced cellular metabolism, growth, and survival (26).In this study, we investigated whether the PI3K/PTEN pathway may be important in regulating Fas-induced apoptosis in type I and type II cells. Indeed, we found a robust correlation between PTEN expression and type I/II Fas-induced apoptosis in a wide variety of cancers. Furthermore, through PTEN gain-of-function and loss-of-function approaches, we demonstrated the ability of the PI3K/PTEN pathway to promote interconversion between the mitochondrially independent type I and mitochondrially dependent type II Fas pathways. Significantly, we found that PTEN haploinsufficiency promotes Bcl-2 sensitivity of Fas-induced apoptosis of primary thymocytes and activation-induced cell death of T lymphocytes. Furthermore, Bcl-2 sensitivity of Fas-induced apoptosis was found to be regulated by PEA-15, in a phosphorylation-dependent manner, and PEA-15 phosphorylation is mediated by the PTEN/PI3K pathway. Thus, our data indicate that the PTEN/PI3K pathway modulates the dependency of cells on the mitochondrial amplification loop to mediate Fas-induced apoptosis and determines whether a cell dies by a type I or type II Fas pathway, in part through regulating PEA-15 activity.     

Seasonal variations of melatonin in ram seminal plasma are correlated to those of testosterone and antioxidant enzymes

Background  

Some breeds of sheep are highly seasonal in terms of reproductive capability, and these changes are regulated by photoperiod and melatonin secretion. These changes affect the reproductive performance of rams, impairing semen quality and modifying hormonal profiles. Also, the antioxidant defence systems seem to be modulated by melatonin secretion, and shows seasonal variations. The aim of this study was to investigate the presence of melatonin and testosterone in ram seminal plasma and their variations between the breeding and non-breeding seasons. In addition, we analyzed the possible correlations between these hormones and the antioxidant enzyme defence system activity.     

Nanometric self-assembling peptide layers maintain adult hepatocyte phenotype in sandwich cultures

Background  

Isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions when cultured. Normally hepatocytes are commonly maintained under limited culture medium supply as well as scaffold thickness. Thus, the cells are forced into metabolic stress that degenerate liver specific functions. This study aims to improve hepatospecific activity by creating a platform based on classical collagen sandwich cultures.