Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Integrative Proteomic Profiling of Protein Activity and Interactions Using Protein Arrays

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.Proteomics is the large-scale analysis of whole proteins and their role in biological systems. Abundance-based proteomics assigns protein functions in normal and pathological processes by quantification of global differences in protein expression levels (1). This classic approach identifies functional biomarkers by comparing samples from healthy individuals and patients. However, this abundance-based approach provides only indirect information about protein function (2). The abundance of a protein is not necessarily correlated with its activity because protein activities are predominantly regulated by a series of post-translational modifications (1, 2). Activity-based proteomics (activity-based protein profiling) is therefore considered an alternative approach to assigning protein functions in biological processes of interest (3). In this approach, specific activity-based probes using fluorescent, radioactive, and affinity tags are usually designed for detection of protein activity (2, 4–6). Activity-based proteomics identifies markers by comparative analyses of activity profiles between healthy and diseased cells and tissues (3, 7, 8). This approach is also used for profiling enzyme inhibitors, for developing therapeutic reagents, and for diagnosis (2, 5). Another functional proteomic approach using large-scale analysis is interactomics or interaction proteomics, which is a useful method for understanding the regulation of proteins in biological systems (9). To elucidate bioactive protein interactions with proteins or ligands, a number of technologies are currently used including the yeast two-hybrid system, affinity purification and mass spectrometry, the protein fragment complementation assay, the luminescence-based mammalian interactome, and protein arrays (9–13). Global differences in the dynamics of the interactome between healthy and diseased individuals provide new insights into causes of disease and can be used for biomarker identification and drug discovery (14–16). Thus, combinatory analyses of abundance, activity, and interaction have great potential in revealing regulation mechanisms and functions of proteins, although such an integrated proteomic approach has not been widely used.These proteomic methods have been coupled with various detection methods including one- or two-dimensional gel electrophoresis, one- or two-dimensional liquid chromatography and tandem mass spectrometry, surface plasmon resonance, and fluorometric assays for analyses of the proteome (9, 11). In combination with specific probes, colorimetric and fluorometric assays using multiwell plates have been extensively used for the determination of the abundance and activity of various proteins. Although often limited by the amount of sample, these methods nonetheless facilitate real-time measurement of changes in protein activity and high-throughput analyses of protein abundances and activities (17). Surface plasmon resonance, a method that does not necessitate labeling of proteins, has also been used for analysis of protein abundance, activity, and binding affinity (18–20). Using only very small amounts of sample, the microarray combined with fluorometric probes is a promising technology for the rapid analysis of a wide variety of biomolecular interactions, protein abundances, and activities. This approach has been used for serodiagnosis and identification of biomarkers by abundance-based protein profiling in human sera (21–25). It has also been used for kinetic studies of carbohydrate-protein (17) and peptide-protein interactions (26, 27). In addition, this technology has been used for the rapid determination of enzyme activities and for the identification of enzyme substrates and inhibitors (24, 28–33). However, combinatory profiling of protein activities and interactions based on array technology has yet to be reported.Using protein arrays, we propose as a model system an integrative proteomic approach for simultaneous profiling of the transamidating activity and interactions of transglutaminase 2 (TG2) with TG2-related proteins. TG2, known as tissue transglutaminase, is a member of the calcium-dependent transglutaminase family. Its activity and interactions are associated with a wide variety of diseases and cellular events (34). TG2 is implicated in the pathogenesis of a wide variety of diseases including inflammatory diseases such as celiac sprue, neurodegenerative disorders such as Huntington''s, Alzheimer''s, and Parkinson''s disease, as well as cancers, cardiovascular diseases, and diabetes (34–36). TG2 is also involved in various cellular events including cell growth, cell differentiation, cell adhesion, extracellular matrix crosslinking, and apoptosis (34, 37, 38). In the present study, the transamidating activity and binding affinity of TG2 for 16 proteins were simultaneously monitored using Cy5-conjugated TG2 and protein arrays (Fig. 1). Using this large-scale analysis, we constructed a quantitative activity-interaction (AI)¹ map to describe the quantitative interaction of TG2 with its related proteins. Thus, this integrative proteomic approach can be used to characterize functions and regulation mechanisms of a target protein in many biological processes of interest.Open in a separate windowFig. 1.Schematic diagram for the simultaneous analysis of transamidating activity and interaction of TG2 with TG2-related proteins. BAPA, 5-(biotinamido)pentylamine; Pr, protein; SA, streptavidin; TG2, transglutaminase 2.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Crowded Cell-like Environment Accelerates the Nucleation Step of Amyloidogenic Protein Misfolding

To understand the role of a crowded physiological environment in the pathogenesis of neurodegenerative diseases, we report the following. 1) The formation of fibrous aggregates of the human Tau fragment Tau-(244–441), when hyperphosphorylated by glycogen synthase kinase-3β, is dramatically facilitated by the addition of crowding agents. 2) Fibril formation of nonphosphorylated Tau-(244–441) is only promoted moderately by macromolecular crowding. 3) Macromolecular crowding dramatically accelerates amyloid formation by human prion protein. A sigmoidal equation has been used to fit these kinetic data, including published data of human α-synuclein, yielding lag times and apparent rate constants for the growth of fibrils for these amyloidogenic proteins. These biochemical data indicate that crowded cell-like environments significantly accelerate the nucleation step of fibril formation of human Tau fragment/human prion protein/human α-synuclein (a significant decrease in the lag time). These results can in principle be predicted based on some known data concerning protein concentration effects on fibril formation both in vitro and in vivo. Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of neurodegenerative diseases by accelerating amyloidogenic protein misfolding and inducing human prion fibril fragmentation, which is considered to be an essential step in prion replication.Amyloid fibrils associated with neurodegenerative diseases such as Alzheimer disease, Parkinson disease, Huntington disease, and transmissible spongiform encephalopathy (TSE)³ (1–5) can be considered biologically relevant failures of the cellular protein quality control mechanisms (6) consisting of molecular chaperones and proteases (7). Up to now, about 20 different proteins with unrelated sequences and tertiary structures are known to form fibrous aggregates associated with various neurodegenerative diseases. These amyloidogenic proteins include both natively unfolded proteins, such as human Tau protein (3) and human α-synuclein (8), and folded globular proteins such as human prion protein (4). There are two faces of protein misfolding in neurodegeneration as follows: a gain of toxic function and a loss of physiological function, which can even occur in combination (9).Human Tau protein, a marker for Alzheimer disease, forms filaments in the brains of patients with Alzheimer disease (3, 10, 11). It has been found that hyperphosphorylation of Tau reduces the binding affinity between Tau and tubulin and contributes to the self-association of Tau and the formation of Tau paired helical filaments (3, 11–13). It has been proposed that glycogen synthase kinase-3β (GSK-3β) hyperphosphorylation of Tau plays an important role in Alzheimer disease (14, 15), and GSK-3β induces an Alzheimer disease-like hyperphosphorylation of Tau when overexpressed in cultured human neurons (16).A large body of data strongly suggests Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, and other TSEs are caused by prions (4). Prions are infectious proteins that can transmit biological information by propagating protein misfolding and aggregation (17). The infectious agent is believed to consist entirely of the prion protein (PrP) and is devoid of nucleic acid (4, 17). Prion biogenesis is associated with the normal protease-sensitive form of the protein (cellular PrP molecule, PrP^C) undergoing structural change into an abnormal, protease-resistant, disease-causing isoform of prion protein (PrP^Sc) (4, 17). Although the mechanism by which PrP^C is converted to PrP^Sc in TSE-infected cells and in vivo is not clear, data from cell-free reactions suggest this process is akin to autocatalytic polymerization (18).Misfolding of Tau and prion proteins has been traditionally and widely studied in dilute solutions (10, 19–21). However, the physiological environment is poorly modeled by such dilute solutions, and biochemical reactions in vivo differ greatly from those in dilute solutions (22). The proteins associated with neurodegenerative diseases form fibrils in a physiological environment crowded with other background macromolecules (22–26), such as proteins, glycosaminoglycans, and proteoglycans (23). Crowding is not confined to cellular interiors but also occurs in the extracellular matrix of tissues (24) and takes place at membrane surfaces (27). For example, blood plasma contains ∼80 g/liter protein, a concentration sufficient to cause significant crowding effects (24). Polysaccharides also contribute to crowding, especially in the extracellular matrix of tissues such as collagen (23, 26). The conversion of PrP from a normal soluble conformation PrP^C to its pathogenic conformation PrP^Sc is believed to occur on the cell surface, in the endocytic vesicles, or in the crowded extracellular matrix (18). Thus, macromolecular crowding on the cell surface and in the extracellular matrix may play an important role in the conformational transition and amyloid formation of PrP in vivo, which have not been fully characterized yet. In vitro, such a crowded environment can be achieved experimentally by adding high concentrations of single or mixed nonspecific crowding agents to the system (23–31). Recently, it has been demonstrated that macromolecular crowding significantly enhances the rate of amyloid formation of α-synuclein (32, 33), amyloid-β peptides (27), and human apolipoprotein C-II (34). However, the role of the crowded physiological environment in the pathogenesis of neurodegenerative diseases is poorly understood so far.To address the contributions of crowded physiological environments on the pathogenesis of neurodegenerative diseases, we report here that macromolecular crowding dramatically accelerates fibril formation by human Tau fragment and by human prion protein under physiological conditions. Our results indicate that macromolecular crowding significantly accelerates the nucleation step of fibril formation of human Tau fragment/human prion protein/human α-synuclein by fitting the data to a sigmoidal equation (35, 36). Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions.     

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics

Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.Targeted protein degradation is an important regulatory mechanism that allows co-ordination of cellular pathways in response to environmental and temporal stimuli (1). The control of diverse biochemical pathways, including cell cycle progression and the response to DNA damage, is mediated, at least in part, by dynamic alterations in protein degradation (2). Previous large scale proteomics studies in mammalian cells have shown that the rate of protein degradation can vary from the timescale of minutes, to essentially infinite stability for metastable proteins (3–8).Most intracellular proteins have similar degradation rates, with a half-life approximating the cell doubling rate. Under 5% of proteins display degradation rates more than threefold faster than the proteome average (3–5, 7). However, degradation rates for individual proteins can change, for example depending on either the cell cycle stage, or signaling events, and can also vary depending on subcellular localization. Disruption of such regulated protein stability underlies the disease mechanisms responsible for forms of cancer, e.g. p53 (9, 10) and the proto-oncogene c-Myc (11).Detection of rapidly degraded proteins can be difficult because of their low abundance. However, advances in mass spectrometry based proteomics have enabled in-depth quantitative analysis of cellular proteomes (12–14). Stable isotope labeling by amino acids in cell culture (SILAC)¹ (15), has been widely used to measure protein properties such as abundance, interactions, modifications, turnover, and subcellular localization under different conditions (16). Subcellular fractionation and protein size separation are also powerful techniques that enhance in-depth analysis of cellular proteomes. Not only do these fractionation techniques increase total proteome coverage, they also provide biological insight regarding how protein behavior differs between subcellular compartments. For example, subcellular fractionation has highlighted differences in the rate of ribosomal protein degradation between the nucleus and cytoplasm, (7, 17). Other studies have also demonstrated the benefit of in-depth subcellular fractionation and created methods for the characterization of how proteomes are localized in organelles (18–20).In this study we have used SILAC-based quantitative mass spectrometry combined with extensive subcellular and protein-level fractionation to identify rapidly degraded proteins in human U2OS cells. We provide a proteome level characterization of a major feedback mechanism involving inhibition of protein translation when the proteasome is inhibited. We also present the Encyclopedia of Proteome Dynamics, a user-friendly online resource providing access to the entire data set.     

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.The CCN (CYR61/Connective Tissue Growth Factor/Nephroblastoma Overexpressed) family of multimodular proteins mediates diverse cellular functions, including cell adhesion, migration, and proliferation (1–3). Overexpression of CCN3, one of the founding members of the family, inhibits proliferation in most types of tumors such as glioblastoma and Ewing sarcoma (4, 5). Similarly, down-regulation of CCN3 has been suggested to promote melanoma progression (6). On the other hand, CCN3 can also promote migration in sarcoma and glioblastoma (4, 7), although a separate study shows that it decreases the invasion of melanoma (6). Therefore, in contrast to its role in growth suppression, the role of CCN3 signaling in cell motility is less clear.Most evidence suggests CCN3 mediates its effects by binding to the integrin proteins, such as the αVβ3 receptors (8, 9), and that CCN3 alters cell adhesion in an integrin-dependent fashion (4, 10). In melanocytes, the discoidin domain receptor 1 mediates CCN3-dependent adhesion (11). CCN3 has also been observed to associate with Notch1 (12), fibulin 1C (13), S100A4 (14), and the gap junction protein Cx43³ (15, 16), suggesting that CCN3 may also modulate cell growth via non-integrin signaling pathways.Gap junction proteins are best known for forming channels between cells, contributing to intercellular communication by allowing the exchange of small ions and molecules (17, 18). Consequently, attenuated intercellular communication has been implicated in promoting carcinogenesis (19, 20). Recent evidence has indicated that connexins can mediate channel-independent growth control through interaction of their C-terminal cytoplasmic tail with various intracellular signaling molecules (21–23). In addition, many Cx43-interacting proteins, including ZO-1 (zonula occludens-1) (24), Drebrin (25), and N-cadherin (26) associate with F-actin, thus placing Cx43 in close proximity to the actin cytoskeleton.In this study, we show for the first time that CCN3 reorganizes the actin cytoskeleton of the breast cancer cells MDA-MB-231 with the formation of multiple cell protrusions, possibly by activating the small GTPase Rac1. Our results also suggest an alternative route by which Cx43 may be functionally linked to actin cytoskeletal signaling via CCN3.