Common Set Of Proteins Research Articles

Transcriptional regulatory network for neuron-glia interactions and its implication for DYT6 dystonia.

Advances in sequencing technologies have identified novel genes associated with inherited forms of dystonia, providing valuable insights into its genetic basis and revealing diverse genetic pathways and mechanisms involved in its pathophysiology. Since identifying genetic variation in the transcription factor coding THAP1 gene linked to isolated dystonia, numerous investigations have employed transcriptomic studies in DYT-THAP1 models to uncover pathogenic molecular mechanisms underlying dystonia. This review examines key findings from transcriptomic studies conducted on in vivo and in vitro DYT-THAP1 models, which demonstrate that the THAP1-regulated transcriptome is diverse and cell-specific, yet it is bound and co-regulated by a common set of proteins. Prominent among its functions, THAP1 and its co-regulatory network target molecular pathways critical for generating myelinating oligodendrocytes that ensheath axons and generate white matter in the central nervous system. Several lines of investigation have demonstrated the importance of myelination and oligodendrogenesis in motor function during development and in adults, emphasizing the non-cell autonomous contributions of glial cells to neural circuits involved in motor function. Further research on the role of myelin abnormalities in motor deficits in DYT6 models will enhance our understanding of axon-glia interactions in dystonia pathophysiology and provide potential therapeutic interventions targeting these pathways.

Mass Spectrometry-Based Proteomic Characterization of Cutaneous Melanoma Ectosomes Reveals the Presence of Cancer-Related Molecules.

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC–MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial–mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.

Characterization of secretomes provides evidence for adipose-derived mesenchymal stromal cells subtypes.

IntroductionThis study was aimed at deciphering the secretome of adipose-derived mesenchymal stromal cells (ADSCs) cultured in standard and hypoxic conditions to reveal proteins, which may be responsible for regenerative action of these cells.MethodsHuman ADSCs were isolated from 10 healthy donors and cultured for 3–4 passages. Cells were serum deprived and cell purity was assessed using multiple cell surface markers. Conditioned media was collected and analyzed using LC-MS with a focus on characterizing secreted proteins.ResultsPurity of the ADSC assessed as CD90+/CD73+/CD105+/CD45-/CD31- cells was greater than 99 % and viability was greater than 97 %. More than 600 secreted proteins were detected in conditioned media of ADSCs. Of these 100 proteins were common to all cultures and included key molecules involved in tissue regeneration such as collagens and collagen maturation enzymes, matrix metalloproteases, matricellular proteins, macrophage-colony stimulating factor and pigment epithelium derived factor. Common set of proteins also included molecules, which contribute to regenerative processes but were not previously associated with ADSCs. These included olfactomedin-like 3, follistatin-like 1 and prosaposin. In addition, ADSCs from the different subjects secreted proteins, which were variable between different cultures. These included proteins with neurotrophic activities, which were not previously associated with ADSCs, such as mesencephalic astrocyte-derived neurotrophic factor, meteorin and neuron derived neurotrophic factor. Hypoxia resulted in secretion of 6 proteins, the most prominent included EGF-like repeats and discoidin I-like domains 3, adrenomedullin and ribonuclease 4 of RNase A family. It also caused the disappearance of 8 proteins, including regulator of osteogenic differentiation cartilage-associated protein.ConclusionsHuman ADSCs with CD90+/CD73+/CD105+/CD45-/CD31-/PDGFRβ+/NG2+/CD146+(−) immunophenotype secrete a large array of proteins, the most represented group is comprised of extracellular matrix components. Number of secreted proteins is largely unaffected by prolonged hypoxia. Variability in the secretion of several proteins from cultured ADSCs of individual subjects suggests that these cells exist as a heterogeneous population containing functionally distinct subtypes, which differ in numbers between donors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0209-8) contains supplementary material, which is available to authorized users.

Neuron-specific protein interactions of Drosophila CASK-β are revealed by mass spectrometry.

Modular scaffolding proteins are designed to have multiple interactors. CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, has been shown to have roles in many tissues, including neurons and epithelia. It is likely that the set of proteins it interacts with is different in each of these diverse tissues. In this study we asked if within the Drosophila central nervous system, there were neuron-specific sets of CASK-interacting proteins. A YFP-tagged CASK-β transgene was expressed in genetically defined subsets of neurons in the Drosophila brain known to be important for CASK function, and proteins present in an anti-GFP immunoprecipitation were identified by mass spectrometry. Each subset of neurons had a distinct set of interacting proteins, suggesting that CASK participates in multiple protein networks and that these networks may be different in different neuronal circuits. One common set of proteins was associated with mitochondria, and we show here that endogenous CASK-β co-purifies with mitochondria. We also determined CASK-β posttranslational modifications for one cell type, supporting the idea that this technique can be used to assess cell- and circuit-specific protein modifications as well as protein interaction networks.

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na+ and Cl− accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na+ and Cl− accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.

Molecular Chaperones as a Common Set of Proteins That Regulate the Invasion Phenotype of Head and Neck Cancer

The goal of this study was to establish a common set of molecules that regulate cell invasion in head and neck cancer (HNC). Five invasive sublines derived from HNC cell lines were established using the Matrigel selection method. Proteomic technology, MetaCore algorithm, and reverse transcriptase-PCR methods were used to search for molecules that contribute to the invasion phenotype. Cellular functional analyses and clinical association studies were applied to examine the significance of the molecules. Fifty-two proteins were identified in more than two of the four independent proteomic experiments, including 10 (19%) molecular chaperones. Seven chaperones were confirmed to be differentially expressed in five sublines, Hsp90α, Hsp90β, Hsp90-B1/Gp96, Hsp70-A5/Grp78, and HYOU1, that upregulate, whereas Hsp60 and glucosidase-α neutral AB (GANAB) downregulate. Four molecules were further investigated. In all cell lines, knockdown of Hsp60 or GANAB and silencing of Gp96 or Grp78 considerably enhanced or reduced cell migration and invasion, respectively. Clinical association studies consistently revealed that low levels of Hsp60 or GANAB and high levels of Gp96 or Grp78 are significantly associated with advanced cancer (P < 0.001 to P = 0.047, respectively, for the four molecules) and poor survival (P < 0.001 to P = 0.025, respectively, for the four molecules). Our study defined molecular chaperones as a common set of proteins that regulate the invasion phenotype of HNC. Loss of the tumor suppression function of Hsp60 or GANAB and acquisition of the oncogenic function of Gp96 or Grp78 contribute to aggressive cancers. These molecules may serve as prognostic markers and targets for cancer drug development.

Abstract 2358: Molecular chaperones as a common set of proteins that regulate the invasion phenotype of head and neck cancer

Abstract Purpose: The goal of this study was to establish a common set of molecules that regulate cell invasion in head and neck cancer (HNC). Experimental design: Five invasive sublines derived from HNC cell lines were established using the Matrigel selection method. Proteomic technology, MetaCore™ algorithm, and RT-PCR methods were used to search for molecules that contribute to the invasion phenotype. Cellular functional analyses and clinical association studies were applied to examine the significance of the molecules. Results: Fifty-two proteins were identified in more than two of the four independent proteomic experiments, including 10 (19%) molecular chaperones. Seven chaperones were confirmed to be differentially expressed in five sublines: Hsp90α, Hsp90β, Hsp90-B1/Gp96, Hsp70-A5/Grp78, HYOU1 up-regulation, while Hsp60 and GANAB down-regulation. Four molecules with the highest differential expressions (&amp;gt; 4-fold) were further investigated. In all cell lines, knockdown of Hsp60 or GANAB and silencing of Gp96 or Grp78 considerably enhanced or reduced cell migration and invasion, respectively. Clinical association studies consistently revealed that low levels of Hsp60 or GANAB and high levels of Gp96 or Grp78 are significantly associated with advanced cancer (P&amp;lt;0.001 to P = 0.047, respectively, for the four molecules) and poor survival (P&amp;lt;0.001 to P=0.025, respectively, for the four molecules). Conclusion: In conclusion, our study defined molecular chaperones as a common set of proteins that regulate the invasion phenotype of head and neck cancer. Loss of the tumor suppression function of Hsp60 or GANAB and acquisition of the oncogenic function of Gp96 or Grp78 contribute to aggressive cancers. These molecules may serve as prognostic markers and targets for cancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2358. doi:10.1158/1538-7445.AM2011-2358

Salt and genotype impact on plant physiology and root proteome variations in tomato

To evaluate the genotypic variation of salt stress response in tomato, physiological analyses and a proteomic approach have been conducted in parallel on four contrasting tomato genotypes. After a 14 d period of salt stress in hydroponic conditions, the genotypes exhibited different responses in terms of plant growth, particularly root growth, foliar accumulation of Na(+), and foliar K/Na ratio. As a whole, Levovil appeared to be the most tolerant genotype while Cervil was the most sensitive one. Roma and Supermarmande exhibited intermediary behaviours. Among the 1300 protein spots reproducibly detected by two-dimensional electrophoresis, 90 exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. A common set of proteins (nine spots), up- or down-regulated by salt-stress whatever the genotype, was detected. But the impact of the tomato genotype on the proteome variations was much higher than the salt effect: 33 spots that were not variable with salt stress varied with the genotype. The remaining number of variable spots (48) exhibited combined effects of the genotype and the salt factors, putatively linked to the degrees of genotype tolerance. The carbon metabolism and energy-related proteins were mainly up-regulated by salt stress and exhibited most-tolerant versus most-sensitive abundance variations. Unexpectedly, some antioxidant and defence proteins were also down-regulated, while some proteins putatively involved in osmoprotectant synthesis and cell wall reinforcement were up-regulated by salt stress mainly in tolerant genotypes. The results showed the effect of 14 d stress on the tomato root proteome and underlined significant genotype differences, suggesting the importance of making use of genetic variability.

Head Module Control of Mediator Interactions

Yeast Mediator proteins interacting with Med17(Srb4) have been expressed at a high level with the use of recombinant baculoviruses and recovered in homogeneous form as a seven subunit, 223 kDa complex. Electron microscopy and single-particle analysis identify this complex as the Mediator head module. The recombinant head module complements "headless" Mediator for the initiation of transcription in vitro. The module interacts with an RNA polymerase II-TFIIF complex, but not with the polymerase or TFIIF alone. This interaction is lost in the presence of a DNA template and associated RNA transcript, recapitulating the release of Mediator that occurs upon the initiation of transcription. Disruption of the head module in a temperature-sensitive mutant in vivo leads to the release of middle and tail modules from a transcriptionally active promoter. The head module evidently controls Mediator-RNA polymerase II and Mediator-promoter interactions.

Regulation of chromosome replication.

The initiation of DNA replication in eukaryotic cells is tightly controlled to ensure that the genome is faithfully duplicated once each cell cycle. Genetic and biochemical studies in several model systems indicate that initiation is mediated by a common set of proteins, present in all eukaryotic species, and that the activities of these proteins are regulated during the cell cycle by specific protein kinases. Here we review the properties of the initiation proteins, their interactions with each other, and with origins of DNA replication. We also describe recent advances in understanding how the regulatory protein kinases control the progress of the initiation reaction. Finally, we describe the checkpoint mechanisms that function to preserve the integrity of the genome when the normal course of genome duplication is perturbed by factors that damage the DNA or inhibit DNA synthesis.

A plant calmodulin-binding motor is part kinesin and part myosin.

Kinesins and myosins are molecular motors that move on microtubules and actin filaments, respectively. These motor proteins are involved in a variety of cellular processes such as intracellular transport, spindle formation and chromosome segregation in eukaryotes (Langford, 1995; Hirokawa, 1998). Although kinesins and myosins hydrolyze ATP to generate force for their movement on cytoskeletal filaments and have similar structural organization (head/motor, stalk and tail) there is virtually no amino acid (aa) sequence identity between the microtubuleand actin-based motors (Kull et al., 1996). We report here that a novel calmodulin-binding protein from plants has domains that are present in microtubuleand actin-based motors, suggesting that this class of plant motors is unique in having kinesin and myosin features. In a protein–protein interaction based screening with calmodulin we recently isolated a cDNA from Arabidopsis encoding a novel calmodulin (CaM)-binding protein, KCBP, with a kinesin-like motor domain at the C-terminus of the protein (Reddy et al., 1996). Motility studies with the C-terminal kinesin-like motor domain alone have shown that it is a minus-end directed microtubule motor (Song et al., 1997). KCBP is unique among all known kinesins and kinesin-like proteins in having a CaMbinding domain (CBD). The homolog of KCBP has been cloned from three other plant systems, suggesting that it is ubiquitous in flowering plants (Narasimhulu and Reddy, 1998). However, a KCBP homolog has not been found in yeast and Caenorhabditis elegans, whose genomes have been completely sequenced, or in any other non-plant systems. In our ongoing analysis of KCBP function, we analyzed KCBP sequence using BLASTP and SMART programs (http://coot.embl-heidelberg.de/SMART) and identified, in addition to kinesin-like motor domain at the C-terminus, a region that is conserved in the tails of some members of myosins (Figure 1). The C-terminal region (amino acids 860–1217) of KCBP showed similarities with the motor region of a large number of microtubulebased motors of which KIFC3, a C-terminal motor, showed the highest sequence similarity (43% identity and 56% similarity, Figure 1A). The tail of myosin VIIa has two long repeats (about ∼460 aa per repeat), each containing a MyTH4 (myosin tail homology 4) domain (∼110 aa) and a talin-like (∼350 aa) domain. Although the MyTH4 domain and the talin-like regions exist singly in some myosins (IV and XII) and in the band 4.1 superfamily (examples include talin and ERM proteins), respectively, the presence of these two regions together (MyTH4+talin-like) occurs only in myosin VIIa (as two repeats) and myosin X (one repeat) (Chen et al., 1996). The N-terminal tail (aa 121–612) of KCBP showed significant similarities to the MyTH4 and talin-like (band 4.1) regions present in myosins VIIa and X. The MyTH4 and the talin-like domains of KCBP showed the highest similarity with the myosin VIIa tail (29% identity and 45% similarity in the MyTH4 domain and 23% identity and 37% similarity in the talin-like region) (Figure 1B). The MyTH4 domain and talin-like region have not been found in any other known members of the kinesin superfamily. Therefore, we conclude that the KCBP is a molecular hybrid consisting of a motor domain from microtubule-based motors and a tail region of actin-based motors (Figure 1C). The significance of MyTH4 and talin-like domains in KCBP and myosins is not known at this time, but the existence of talin-like region together with MyTH4 domain in KCBP, myosin VIIa and X is interesting and points to some functional significance. Based on what is known about KCBP and myosin VIIa, we propose that these domains may be involved in one or more of the following functions. One possibility is that the KCBP and myosin VIIa-like proteins could interact with some unknown common protein(s) through their tail homology regions to either cross-bridge microtubule and actin filaments or facilitate cargo exchange between these two types of molecular motors. An elegant study by Lille and Brown (1992) showed that a lethal mutation resulting from a defect in a myosin motor could be complemented by a kinesin motor protein which also suggests that actinand microtubule-motors are likely to interact with some common set of proteins. Furthermore,

Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes.

cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.

Common set of proteins in Aplysia sensory neurons affected by an in vitro analogue of long-term sensitization training, 5-HT and cAMP

An in vitro analogue of long-term behavioral training in Aplysia was developed to simulate the intensity and timing of shocks delivered to the body wall of animals during sensitization training. The in vitro training analogue consisted of electrical stimulation of peripheral nerves of isolated pleural-pedal ganglia. We found that the in vitro training analogue led to long-term (24 h) changes in the membrane currents of sensory neurons; these changes were similar to those produced by behavioral training. Using two dimensional polyacrylamide gel electrophoresis, we examined early effects on protein synthesis in pleural sensory neurons induced by the training analogue. Incorporation of amino acid into 5 proteins was affected at the end of training. Incorporation of amino acid into some of these proteins was also affected by serotonin (5-HT) and by an analogue of cyclic adenosine monophosphate (cAMP). These results suggest that the effects on protein synthesis that are produced by the in vitro training analogue are mediated, at least in part, by release of 5-HT and an increase in the level of cAMP in the sensory neurons.

Thrombin and collagen induce rapid phosphorylation of a common set of cellular proteins on tyrosine in human platelets

The ability of thrombin and collagen to induce protein-tyrosine phosphorylation in intact human platelets was assessed by using antibodies to phosphotyrosine in conjugation with immunoblots. Upon stimulation by thrombin there was an increase in the amount of protein-tyrosine phosphorylation of three bands with molecular masses of 135, 124, and 76 kDa in a time-dependent manner. The tyrosine phosphorylation in these three proteins increased in a concurrent fashion and reached a maximum level in 10 s and then a plateau or a slight decrease. Stimulation by collagen was also followed by an increase in tyrosine phosphorylation of 135- and 124-kDa proteins. Unlike stimulation by thrombin, collagen induced no obvious tyrosine phosphorylation of 76-kDa protein. The time courses for thrombin- or collagen-induced protein-tyrosine phosphorylation were similar to that for [14C] serotonin release. These results suggest that 135- and 124-kDa proteins are a common set of proteins that become phosphorylated on tyrosine residue during platelet activation.