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PubMed1990

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor.

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Amino Acid Sequence↗
PubMed1995

B-50 (GAP-43) immunoreactivity is rarely detected within intact catecholaminergic and serotonergic axons innervating the brain and spinal cord of the adult rat, but is associated with these axons following lesion.

The persistence of high levels of B-50 (GAP-43) in fibers innervating various regions of the adult central nervous system is generally thought to characterize neuronal systems capable of undergoing morphological plasticity. In a recent series of in situ hybridization studies, it has been shown that most catecholaminergic and serotonergic neurons of the adult rat brain express high levels of B-50 mRNA. The present study addresses the question whether high expression of B-50 mRNA in the catecholaminergic and serotonergic perikarya corresponds with detectable high levels of the B-50 protein in the efferent axonal fibers that innervate various regions of the adult rat brain and spinal cord. For this purpose, vibratome sections were doubly immunostained for B-50 and for tyrosine hydroxylase or serotonin and were analyzed by laser scanning confocal microscope. Colocalizations were investigated either (1) in regions of intact rat brain and spinal cord in which particular concentrations of B-50 immunoreactive fibers appeared codistributed with catecholaminergic or serotonergic fibers or (2) in intrahypothalamic portions of the medial forebrain bundle in which a surgical lesion was made. In the intact brain, frequent colocalizations of B-50 and tyrosine hydroxylase were detected in fibers innervating both the mediobasal hypothalamus and the neurointermediate hypophysial lobe. In all the other regions examined, the analysis of thin optical sections demonstrated that immunoreactivity to B-50 was only rarely associated with axonal profiles immunoreactive to tyrosine hydroxylase or to serotonin. By contrast, in the lesioned medial forebrain bundle B-50 immunoreactivity was found to be associated with numerous catecholaminergic and serotonergic axonal sprouts that regenerate around the surgical lesion. These data indicate that the majority of intact catecholaminergic and serotonergic axons innervating the adult rat brain and spinal cord contains low levels of B-50. However, following axotomy, B-50 is immunocytochemically detectable in the regenerating sprouts produced by both types of axonal fibers. This suggests that under basal conditions the relatively high content of B-50 mRNA in monoaminergic perikarya does not lead to appreciable accumulation of B-50 within corresponding axonal fibers and terminals, whereas conditions of morphological reorganization induce increased production of B-50 that accumulates within monoaminergic axonal sprouts.

Animals↗
PubMed1987

[Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond].

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.

Carbohydrate Conformation↗
PubMedDate not supplied

Distribution and co-localization of calbindin D28k with VIP and neuropeptide Y but not somatostatin, galanin and substance P in the enteric nervous system of the rat.

Calbindin D28k, previously demonstrated in the mammalian central nervous system, has been localized to discrete neurons in the enteric nervous system of the rat. Calbindin D28k is present in cell bodies in both the myenteric and submucous plexi and in interganglionic nerve fibers in all regions of the gastrointestinal tract. Immunoreactive nerve fibers were also detected in the mucosal region, although none were observed in the pyloric sphincter, circular or longitudinal muscle layers. The highest concentration of immunoreactivity was present in the submucosal plexus and mucosa of the colon. Western blot analysis of the protein detected by the antiserum confirmed that it comigrated with purified calbindin D28k and the single immunoreactive band seen in extracts from rat brain. The colocalization of calbindin D28k with components of the peptidergic innervation was also investigated. Of the peptides studied the neurons containing both vasoactive intestinal polypeptide and neuropeptide Y in the submucous plexus were seen to exhibit calbindin D28k immunoreactivity. The neurons containing somatostatin, galanin and substance P did not demonstrate co-localization. In the stomach, calbindin D28k was detected within a small number of epithelial cells which were found to correspond to a sub-population of the somatostatin-immunoreactive endocrine cells.

Animals↗
PubMed1995

Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose.

Calcium mobilization from intracellular pools couples many stimuli to responses in plant cells. Cyclic adenosine 5'-diphosphoribose (cADPR), which interacts with a ryanodine receptor in certain animal cells, was shown to elicit calcium release at the vacuolar membrane of beet storage root. The vacuolar calcium release pathway showed similarities to cADPR-gated calcium release in animal cells, including inhibition by ruthenium red, ryanodine activation, and high affinity for cADPR [Michaelis constant (Km) = 24 +/- 7 nanomolar]. Analysis by patch-clamping demonstrated that the cADPR-gated pathway in beet is voltage-dependent over the physiological range, does not spontaneously desensitize, and is colocalized with an inositol 1,4,5-trisphosphate (InsP3)-gated calcium release pathway in individual vacuoles.

Adenosine Diphosphate Ribose↗
PubMed2026

FANCI promotes esophageal squamous cell carcinoma progression and cell cycle regulation and interacts with FANCD2.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor clinical outcomes, and reliable molecular biomarkers and therapeutic targets remain limited. Fanconi anemia group I protein (FANCI) is a core component of the Fanconi anemia (FA) pathway, but its expression pattern, clinical significance, and functional role in ESCC have not been comprehensively defined. This study aimed to investigate FANCI expression and prognostic value in ESCC, assess its effects on malignant cellular phenotypes and tumor growth, and explore its potential mechanistic relationship with Fanconi anemia group D2 protein (FANCD2) and cell-cycle regulation. METHODS: Multi-cohort analyses were performed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, together with ESCC single-cell RNA sequencing (RNA-seq) data. FANCI functions were assessed by bidirectional gain- and loss-of-function experiments in vitro (proliferation, colony formation, migration, invasion, apoptosis, and cell-cycle assays) and by xenograft models in vivo. Mechanistic studies included protein-protein interaction (PPI) analyses, co-immunoprecipitation (Co-IP), and immunofluorescence (IF) colocalization. RESULTS: FANCI was consistently upregulated in ESCC across bulk transcriptomic datasets and was further supported by quantitative polymerase chain reaction (qPCR), Western blotting, and immunohistochemistry (IHC). FANCI discriminated ESCC from normal tissues in TCGA-ESCC and was independently validated in GSE53624 [area under the curve (AUC) =0.940 and 0.975, respectively]. FANCI was associated with poorer overall survival (OS) and shorter disease-free interval (DFI), and these findings were validated in an independent GEO cohort. Functionally, FANCI promoted ESCC cell proliferation, migration, and invasion, while inhibiting apoptosis; FANCI knockdown suppressed tumor growth in vivo and induced G2/M cell-cycle arrest. Mechanistically, FANCI physically interacted with FANCD2, colocalized with FANCD2 in the nucleus, and was associated with altered FANCD2 protein abundance, consistent with cell-cycle and DNA repair-related programs. Single-cell analysis indicated that FANCI was enriched in epithelial cells and associated with higher activity of malignant functional programs. In TCGA-ESCC, FANCI-high tumors showed distinct mutation profiles, a trend toward increased tumor mutation burden (TMB), and altered immune-associated signatures. CONCLUSIONS: FANCI is upregulated in ESCC and is associated with diagnostic and prognostic value. It promotes malignant phenotypes and tumor growth, potentially through a FANCI-FANCD2-linked cell-cycle/DNA repair program, supporting FANCI as a candidate biomarker and therapeutic target in ESCC.

Esophageal squamous cell carcinoma (ESCC)↗
PubMed1990

Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves.

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.

Animals↗
PubMed1993

Receptor-mediated endocytosis of alpha 2 macroglobulin by a glioma cell line.

Previous experiments have shown that human neoplastic and embryonic glial cell lines synthesize and secrete in culture, alpha 2 macroglobulin (alpha 2M), a broad spectrum proteinase inhibitor present in serum and extracellular fluids. The present study was aimed to investigate the presence of alpha 2M receptors on glial cell membrane, since several non-neural cell types producing alpha 2M also express alpha 2M receptors. By flow cytometric analysis, immunofluorescence and immunoelectronmicroscopy techniques we demonstrate an alpha 2M receptor-related immunoreactivity on the plasma membrane of a human glioma cell line. Ultrastructural experiments reveal a close colocalization of immunoreactivities for alpha 2M and its receptor in clathrin-coated pits and vesicles, structures typically involved in receptor-mediated endocytic pathways.

Endocytosis↗
PubMed1995

The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures.

The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of human papillomavirus E4 proteins: identification of structural features important in the formation of cytoplasmic E4/cytokeratin networks in epithelial cells.

We have previously demonstrated that human papillomavirus type 1 (HPV 1) and 16 (HPV 16) E4 proteins form cytoplasmic filamentous networks which specifically colocalize with cytokeratin intermediate-filament (IF) networks when expressed in simian virus 40-transformed keratinocytes. The HPV 16 (but not the HPV 1) E4 protein induced the collapse of the cytokeratin networks. (S. Roberts, I. Ashmole, G. D. Johnson, J. W. Kreider, and P. H. Gallimore, Virology 197:176-187, 1993). The mode of interaction of E4 with the cytokeratin IFs is unknown. To identify E4 sequences important in mediating this interaction, we have constructed a large panel of mutant HPV (primarily HPV 1) E4 proteins and expressed them by using the same simian virus 40-epithelial expression system. Mutation of HPV 1 E4 residues 10 to 14 (LLGLL) abrogated the formation of cytoplasmic filamentous networks. This sequence corresponds to a conserved motif, LLXLL, found at the N terminus of other E4 proteins, and similar results were obtained on deletion of the HPV 16 motif, LLKLL (residues 12 to 16). Our findings indicate that this conserved motif is likely to play a central role in the association between E4 and the cytokeratins. An HPV 1 E4 mutant protein containing a deletion of residues 110 to 115 induced the collapse of the cytokeratin IFs in a manner analogous to the HPV 16 E4 protein. The sequence deleted, DLDDFC, is highly conserved between cutaneous E4 proteins. HPV 1 E4 residues 42 to 80, which are rich in charged amino acids, appeared to be important in the cytoplasmic localization of E4. In addition, we have mapped the N-terminal residues of HPV 1 E4 16-kDa and 10/11-kDa polypeptides expressed by using the baculovirus system and shown that they begin at tyrosine 16 and alanine 59, respectively. Similar-sized E4 proteins are also found in vivo. N-terminal deletion proteins, which closely resemble the 16-kDa and 10/11-kDa species, expressed in keratinocytes were both cytoplasmic and nuclear but did not form cytoplasmic filamentous networks. These findings support the postulate that N-terminal proteolytic processing of the E1-- E4 protein may modulate its function in vivo.

Adult↗
PubMed1989

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver.

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.

Animals↗
PubMed1992

PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.

Cytoskeleton↗
PubMed1995

Neuropeptides phase shift the mammalian circadian pacemaker.

We studied the influence on circadian rhythms of peptides that have been reported to be colocalized in suprachiasmatic nucleus (SCN) neurons. Gastrin-releasing peptide (GRP1-27), peptide histidine isoleucine (PHI), and vasoactive intestinal polypeptide (VIP) were microinjected into the suprachiasmatic nucleus (SCN) region of Syrian hamsters free running under three different constant lighting conditions. All peptide injections caused phase-dependent phase shifts of hamster locomotor activity rhythms which were unaffected by constant lighting conditions. GRP1-27 (150 pmol) caused large phase delays when injected at circadian times (CT) 12-16, modest phase advances when administered at CT20-24, and few shifts during the subjective day. Injections of saline vehicle at any of these phases caused only very small phase shifts. Phase delays induced by GRP1-27 at CT12-14 were dose dependent, unrelated to injection volume (at a constant dose), and attenuated by pretreatment with the BN/GRP-preferring receptor antagonist BIM 26226. VIP (150 pmol) caused moderate phase delays at CT12-14 and moderate phase advances at CT20-24. PHI (150 pmol) caused moderate phase delays at CT12-14 only. Coadministration of 150 pmol of GRP1-27, PHI, and VIP in an equimolar neuropeptide cocktail (50 pmol of each peptide) caused phase delays at CT12-14 and phase advances at CT20-24 which did not differ from those induced by 150 pmol of GRP1-27 alone at these phases. The shifts induced by 150 pmol of the peptide cocktail were smaller than the sum of the shifts induced by 50 pmol doses of each peptide administered separately at those phases. Since the phase-delaying effects of the cocktail were weaker than the summed effects of the component 50 pmol doses of the peptides, these data demonstrate a lack of synergism among the effects of these peptides. Since GRP1-27 (150 pmol) evoked shifts similar in magnitude to those of the cocktail, there is no evidence that these apparently colocalized neuropeptides must interact to exert maximal effects on the circadian pacemaker.

Analysis of Variance↗
PubMed1992

Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton.

Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847-5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2-terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine-rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Enhanced E-cadherin expression in epidermal growth factor receptor expressing cells.

Expression of the epidermal growth factor receptor (EGFr) and the cell-cell adhesion molecule E-cadherin have individually been implicated in the biological activity of the most common human malignancies. There is also evidence for colocalization and for a correlation in the expression of these two proteins in human cells. To better define the relationship between these two gene products, we used immunohistochemistry and Western blot analysis to compare E-cadherin expression in various well characterized cell lines lacking expression of EGFr or expressing wild type, functional mutant or non functioning mutant EGFr. Parental NR6 cells, which lack endogenous EGFr, and a derivitive cell line NR6M721, which expresses EGFr lacking tyrosine kinase activity, showed low levels of E-cadherin expression with or without stimulation with EGF. In contrast, the derivative NR6c'973 cell line, which expresses an active EGFr defective in EGF induced internalization and down-regulation and NR6 cells expressing wild type EGFr showed strong E-cadherin expression. These results suggest that EGFr activation may regulate or enhance E-cadherin expression.

3T3 Cells↗
PubMed1992

Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus.

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.

Adenosine Triphosphatases↗
PubMed2026

Systematic Identification of Therapeutic Targets and Repurposed Drugs for Stroke: From Genome Causal Analysis to Multilevel Validation.

BACKGROUND: Stroke is a severe cerebrovascular disease characterized by narrow time windows and complications. This study aimed to identify novel drug targets and repurposed drugs for stroke. METHODS: This study used expression quantitative trait loci data from druggable genes in brain and blood as instrumental variables. Mendelian randomization, colocalization, and phenome-wide Mendelian randomization were applied to evaluate causal relationships and potential side effects, with stroke and ischemic stroke as primary outcomes. Preclinical validation used oxygen-glucose deprivation/reperfusion and middle cerebral artery occlusion/reperfusion models. Pharmacological and behavioral assessments evaluated the therapeutic potential of candidate targets and drugs. Additionally, proteomic sequencing was performed following GGCX (γ-glutamyl carboxylase) overexpression to explore its biological functions. RESULTS: Elevated GGCX expression in brain and blood was potentially causally associated with reduced risk of stroke and ischemic stroke, supported by colocalization evidence, although potential cardiovascular risks could not be excluded. Drug repositioning identified ifenprodil as a candidate agent that reduced infarction volume, improved motor and cognitive functions, and reversed GGCX downregulation in mice. Ifenprodil treatment and GGCX overexpression alleviated oxygen-glucose deprivation/reperfusion-induced injury and upregulated GGCX expression. Mechanistically, GGCX conferred neuroprotection by regulating protein homeostasis, suppressing inflammation, promoting metabolic recovery, and modulating nuclear transcriptional regulation. CONCLUSIONS: This study established a potential causal link between GGCX and stroke risk, particularly ischemic stroke. GGCX represents a promising therapeutic target for ischemic stroke. Targeted GGCX expression upregulation and drug repurposing, particularly ifenprodil, may offer novel therapeutic avenues. Further validation is warranted to assess clinical efficacy and safety.

Animals↗
PubMed1991

The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine-containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.

Animals↗