Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex.

Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP downregulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this data set identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating.

Intellectual abilities in tuberous sclerosis complex: risk factors and correlates from the Tuberous Sclerosis 2000 Study.

BACKGROUND: Tuberous sclerosis complex (TSC) is associated with intellectual disability, but the risk pathways are poorly understood. METHOD: The Tuberous Sclerosis 2000 Study is a prospective longitudinal study of the natural history of TSC. One hundred and twenty-five UK children age 0-16 years with TSC and born between January 2001 and December 2006 were studied. Intelligence was assessed using standardized measures at ≥2 years of age. The age of onset of epilepsy, the type of seizure disorder, the frequency and duration of seizures, as well as the response to treatment was assessed at interview and by review of medical records. The severity of epilepsy in the early years was estimated using the E-Chess score. Genetic studies identified the mutations and the number of cortical tubers was determined from brain scans. RESULTS: TSC2 mutations were associated with significantly higher cortical tuber count than TSC1 mutations. The extent of brain involvement, as indexed by cortical tuber count, was associated with an earlier age of onset and severity of epilepsy. In turn, the severity of epilepsy was strongly associated with the degree of intellectual impairment. Structural equation modelling supported a causal pathway from genetic abnormality to cortical tuber count to epilepsy severity to intellectual outcome. Infantile spasms and status epilepticus were important contributors to seizure severity. CONCLUSIONS: The findings support the proposition that severe, early onset epilepsy may impair intellectual development in TSC and highlight the potential importance of early, prompt and effective treatment or prevention of epilepsy in tuberous sclerosis.

BMK1 is involved in the regulation of p53 through disrupting the PML-MDM2 interaction.

Promyelocytic leukemia protein (PML) modulates the p53 tumor suppressor through its interaction with p53 and MDM2. We found that activated big MAP kinase 1 (BMK1) preferentially associates with PML isoform IV and disrupts PML-MDM2 interaction. Doxorubicin, a common chemotherapeutic agent, is known to promote PML-mediated p53 activation in part by promoting PML-dependent MDM2 nucleolar sequestration. We discovered that BMK1 deactivation coupled with doxorubicin synergistically enhanced MDM2 nucleolar sequestration and, consequently, promoted PML-mediated p53 upregulation leading to tumor cell apoptosis in vitro and tumor regression in vivo. Collectively, these results not only suggest that BMK1 activity has a role in suppressing p53 by blocking the interaction between PML and MDM2, but also implicate that pharmacological BMK1 inhibitor should significantly enhance the anticancer capacity of doxorubicin-based chemotherapy.

Scientific frontiers: emerging technologies for salivary diagnostics.

Saliva, a biofluid historically well-studied biochemically and physiologically, has entered the post-genomic 'omics' era, where its proteomic, genomic, and microbiome constituents have been comprehensively deciphered. The translational path of these salivary constituents has begun toward a variety of personalized individual medical applications, including early detection of cancer. Salivary diagnostics is a late-comer, but it is catching up where dedicated resources, like the Salivaomics Knowledge Base (SKB), now have taken center stage in the dissemination of the diagnostic potentials of salivary biomarkers and other translational and clinical utilities.

Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling.

Deregulated Her2/ErbB2 receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study innate cellular mechanisms for containing Her2 oncogenic phosphorylation, a siRNA phosphatase library was screened for cellular phosphatase(s) that enhance phosphorylation in the signaling motif of Her2 after knockdown. We found that silencing protein tyrosine phosphatase PTPN13 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain and promoted the invasiveness of Her2-deregulated cancer cells. In addition, we discovered that growth factor-induced phosphorylation of PTPN13 was essential for the dephosphorylation of Her2 suggesting a negative feedback mechanism induced by growth factor to inhibit cellular Her2 activity through PTPN13. Importantly, we showed that PTPN13 mutations previously reported in human tumors significantly reduced the phosphatase activity of PTPN13, and consequently elevated the oncogenic potential of Her2 and the invasiveness of Her2-overexpressing human cancer cells. Taken together, these results suggest that cellular PTPN13 inhibits Her2 activity by dephosphorylating the signal domain of Her2 and plays a role in attenuating invasiveness and metastasis of Her2 overactive tumors.

Age related macular degeneration and sun exposure, iris colour, and skin sensitivity to sunlight.

BACKGROUND/AIM: It has been suggested that sun exposure may be a risk factor for age related macular degeneration (AMD) and that skin sensitivity to sunlight and iris colour could be confounding factors. The aim was to investigate this further in the white population. METHODS: 446 cases with end stage AMD were compared with 283 spouse controls. Data on sun exposure, places of residence, iris colour, subjective assessment of change in iris colour, hair colour at age 20, and skin sensitivity were obtained using a questionnaire. Iris colour was graded clinically by comparison with standard photographs. AMD was graded using stereoscopic colour fundus photographs as well as clinical examination and was defined as the presence of geographic atrophy or choroidal neovascularisation. All variables were included in a multiple logistic regression model including age, sex, and smoking. RESULTS: There was no association between AMD and sun exposure or related factors except for the suggestion of an association between sunburn prone skin type and geographic atrophy which reached borderline significance. CONCLUSIONS: No significant association between AMD and sun exposure, iris colour, change in iris colour, or hair colour was demonstrated.

Smoking and age related macular degeneration: the number of pack years of cigarette smoking is a major determinant of risk for both geographic atrophy and choroidal neovascularisation.

BACKGROUND/AIMS: There is evidence that smoking is a risk factor for age related macular degeneration (AMD). However, not all studies have demonstrated this association and several key questions about the role of smoking in AMD have still to be determined. The aim of this study was to further investigate this relation for both choroidal neovascularisation (CNV) and geographic atrophy (GA). METHODS: To investigate the relation between smoking and the risk of developing age related macular degeneration (AMD) in white people, 435 cases with end stage AMD were compared with 280 controls. All subjects had graded stereoscopic colour fundus photography and AMD was defined as the presence of GA or CNV. Smoking history was assessed using multiple parameters in a detailed questionnaire. RESULTS: Comparison of current and former smokers with non-smokers was consistent with smoking being a risk factor for AMD but did not reach statistical significance. There was a strong association between AMD and pack years of cigarette smoking (p = 0.002), the odds ratio increasing with the amount smoked; for subjects with more than 40 pack years of smoking the odds ratio was 2.75 (95% CI 1.22 to 6.20) compared with non-smokers. Both types of AMD showed a similar relation; smoking more than 40 pack years of cigarettes was associated with an odds ratio of 3.43 (95% CI 1.28 to 9.20) for GA and 2.49 (95% CI 1.06 to 5.82) for CNV. Stopping smoking was associated with reduced odds of AMD and the risk in those who had not smoked for over 20 years was comparable to non-smokers. The risk profile was similar for males and females. Passive smoking exposure was associated with an increased risk of AMD (OR 1.87; 95% CI 1.03 to 3.40) in non-smokers. CONCLUSIONS: The authors have demonstrated a strong association between the risk of both GA and CNV and pack years of cigarette smoking. This provides support for a causal relation between smoking and AMD. They also show an increased risk for AMD in non-smokers exposed to passive smoking. Stopping smoking appears to reduce the risk of developing AMD.

Tuberous sclerosis presenting in late adult life.

A 59 year old woman presented with a three year history of left sided weakness. Magnetic resonance imaging of the brain showed a large high signal lesion occupying most of the right temporal lobe with mass effect. A probable diagnosis of low grade glioma led to temporal lobectomy. Histology revealed dysplastic cortical morphology typical of tuberous sclerosis. There were no clinical signs or family history of the disease. Ultrasound showed multiple bilateral renal angiomyolipomas, confirming the diagnosis of tuberous sclerosis. Molecular genetic analysis of peripheral white blood cells identified a novel mis-sense mutation R1409W in exon 33 of the TSC2 gene.

Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk.

Selective degradation of single subunits of multimeric complexes by the ubiquitin pathway underlies multiple regulatory switches, including those involving cyclins and Cdk inhibitors. The machinery that segregates ubiquitinated proteins from unmodified partners prior to degradation remains undefined. We report that ubiquitinated Sic1 (Ub-Sic1) embedded within inactive S phase cyclin-Cdk (S-Cdk) complexes was rapidly degraded by purified 26S proteasomes, yielding active S-Cdk. Mutant proteasomes that failed to degrade Ub-Sic1 activated S-Cdk only partially in an ATP-dependent manner. Whereas Ub-Sic1 was degraded within approximately 2 min, spontaneous dissociation of Ub-Sic1 from S-Cdk was approximately 200-fold slower. We propose that the 26S proteasome has the intrinsic capability to extract, unfold, and degrade ubiquitinated proteins while releasing bound partners untouched. Activation of S-Cdk reported herein represents a complete reconstitution of the regulatory switch underlying the G1/S transition in budding yeast.

p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation.

p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.

In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses.

We report a strategy for high through-put sequence analyses of large MHC class II-bound peptide repertoires which combines automated electrospray ionization tandem mass-spectrometry with computer-assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I-Ab in activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H-2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II-bound self peptides derived from cytosolic proteins exists in vivo.

A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Implication of a novel multiprotein Dam1p complex in outer kinetochore function.

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells.

The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.

Retinoschisin, the X-linked retinoschisis protein, is a secreted photoreceptor protein, and is expressed and released by Weri-Rb1 cells.

X-linked retinoschisis is characterized by microcystic-like changes of the macular region and schisis within the inner retinal layers, leading to visual deterioration in males. Many missense and protein-truncating mutations of the causative gene RS1 have now been identified and are thought to be inactivating. RS1 encodes a 224 amino acid protein, retinoschisin, which contains a discoidin domain but is of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within retinoschisin, which detects a protein of approximately 28 kDa in retinal samples reduced with dithiothreitol, but multimers sized >40 kDa under non-reducing conditions. A screen of human tissues with this antibody reveals retinoschisin to be retina specific and the antibody detects a protein of similar size in bovine and murine retinae. We investigated the expression pattern in the retina of both RS1 mRNA (using in situ hybridization with riboprobes) and retinoschisin (using immunohistochemistry). The antisense riboprobe detected RS1 mRNA only in the photoreceptor layer but the protein product of the gene was present both in the photoreceptors and within the inner portions of the retina. Furthermore, differentiated retinoblastoma cells (Weri-Rb1 cells) were found to express RS1 mRNA and to release retinoschisin. These results suggest that retinoschisin is released by photo-receptors and has functions within the inner retinal layers. Thus, X-linked retinoschisis is caused by abnormalities in a putative secreted photoreceptor protein and is the first example of a secreted photo-receptor protein associated with a retinal dystrophy.

Genetic susceptibility to age related macular degeneration.

Age related macular degeneration (AMD) is the leading cause of visual impairment in the elderly and a major cause of blindness in the developed world. The disease can take two forms, geographic atrophy and choroidal neovascularisation. The pathogenesis of AMD is poorly understood. There are undoubtedly environmental and other risk factors involved and the adverse effect of smoking is well established. Several studies have shown that genetic factors are important but leave uncertainty about the magnitude and nature of the genetic component and whether it varies with the type of AMD. Several hereditary retinal dystrophies show similarities to AMD and these genes are potential candidate susceptibility genes. Particular interest has focused on the ABCR gene which is responsible for autosomal recessive Stargardt macular dystrophy. It has been claimed that heterozygotes for ABCR mutations are predisposed to AMD but the data are conflicting. Studies of the genes responsible for autosomal dominant Sorsby fundus dystrophy, Doyne honeycomb retinal dystrophy, and Best disease have given negative results. In one large AMD family, linkage has been reported to markers in 1q25-q31. Recent data suggest that the ApoE epsilon4 allele may be associated with reduced risk of AMD. A better understanding of the genetic factors in AMD would contribute to understanding the pathogenesis. If those at risk could be identified it may be possible to modify lifestyle or develop novel therapies in the presymptomatic stage to prevent disease or decrease its severity.

Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells.

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is upregulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.

ATP and the core "alpha-Crystallin" domain of the small heat-shock protein alphaB-crystallin.

Electrospray ionization mass spectrometry (ESI-LC/MS) of tryptic digests of human alphaB-crystallin in the presence and absence of ATP identified four residues located within the core "alpha-crystallin" domain, Lys(82), Lys(103), Arg(116), and Arg(123), that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human alphaB-crystallin produced in the presence and absence of ATP were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven chymotryptic cleavage sites, Trp(60), Phe(61), Phe(75), Phe(84), Phe(113), Phe(118), and Tyr(122), located near or within the core alpha-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K(m) for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core alpha-crystallin domain conserved in nearly all identified small heat-shock proteins that act as molecular chaperones.