Orthogonal IMiD-Degron Pairs Induce Selective Protein Degradation in Cells.

Immunomodulatory imide drugs (IMiDs) including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short zinc finger (ZF) degron motif. These IMiDs, however, also induce degradation of endogenous neosubstrates, including IKZF1 and IKZF3. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogs against 8380 ZF mutants. This yielded a bumped IMiD analog that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1 and IKZF3. In proof-of-concept studies, this system was applied to induce efficient degradation of TRIM28, a disease-relevant protein with no known small molecule binders. We anticipate that this system will make a valuable addition to the current arsenal of degron systems for use in target validation.

Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway.

The Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Previously, YAP was proposed to inhibit skeletal size by repressing chondrocyte proliferation and differentiation. We find that, in vitro, Yap/Taz double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear nls-YAP5SA accelerates proliferation, in line with the canonical role of this pathway in most tissues. However, in vivo, cartilage-specific knockout of Yap/Taz does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. Cartilage-specific expression of nls-YAP5SA or knockout of Lats1/2 do not increase cartilage growth, but instead lead to catastrophic malformations resembling chondrodysplasia or achondrogenesis. Physiological YAP target genes in cartilage include Ctgf, Cyr61 and several matrix remodelling enzymes. Thus, YAP/TAZ activity controls chondrocyte proliferation in vitro, possibly reflecting a regenerative response, but is dispensable for chondrocyte proliferation in vivo, and instead functions to control cartilage morphogenesis via regulation of the extracellular matrix.

MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival.

A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest.

The Topo2a-dependent arrest is associated with faithful segregation of sister chromatids and has been identified as dysfunctional in numerous tumour cell lines. This genome-protecting pathway is poorly understood and its characterization is of significant interest, potentially offering interventional opportunities in relation to synthetic lethal behaviours in arrest-defective tumours. Using the catalytic Topo2a inhibitor ICRF193, we have performed a genome-wide siRNA screen in arrest-competent, non-transformed cells, to identify genes essential for this arrest mechanism. In addition, we have counter-screened several DNA-damaging agents and demonstrate that the Topo2a-dependent arrest is genetically distinct from DNA damage checkpoints. We identify the components of the SMC5/6 complex, including the activity of the E3 SUMO ligase NSE2, as non-redundant players that control the timing of the Topo2a-dependent arrest in G2. We have independently verified the NSE2 requirement in fibroblasts from patients with germline mutations that cause severely reduced levels of NSE2. Through imaging Topo2a-dependent G2 arrested cells, an increased interaction between Topo2a and NSE2 is observed at PML bodies, which are known SUMOylation hotspots. We demonstrate that Topo2a is SUMOylated in an ICRF193-dependent manner by NSE2 at a novel non-canonical site (K1520) and that K1520 sumoylation is required for chromosome segregation but not the G2 arrest.

The Dynamics of TGF-ß Signaling Are Dictated by Receptor Trafficking via the ESCRT Machinery.

Signal transduction pathways stimulated by secreted growth factors are tightly regulated at multiple levels between the cell surface and the nucleus. The trafficking of cell surface receptors is emerging as a key step for regulating appropriate cellular responses, with perturbations in this process contributing to human diseases, including cancer. For receptors recognizing ligands of the transforming growth factor ß (TGF-ß) family, little is known about how trafficking is regulated or how this shapes signaling dynamics. Here, using whole genome small interfering RNA (siRNA) screens, we have identified the ESCRT (endosomal sorting complex required for transport) machinery as a crucial determinant of signal duration. Downregulation of ESCRT components increases the outputs of TGF-ß signaling and sensitizes cells to low doses of ligand in their microenvironment. This sensitization drives an epithelial-to-mesenchymal transition (EMT) in response to low doses of ligand, and we demonstrate a link between downregulation of the ESCRT machinery and cancer survival.

Multiomic Analysis of the UV-Induced DNA Damage Response.

In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response.

Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.

A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.

Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer.

Activating mutations in the EGF receptor (EGFR) are associated with clinical responsiveness to EGFR tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib. However, resistance eventually arises, often due to a second EGFR mutation, most commonly T790M. Through a genome-wide siRNA screen in a human lung cancer cell line and analyses of murine mutant EGFR-driven lung adenocarcinomas, we found that erlotinib resistance was associated with reduced expression of neurofibromin, the RAS GTPase-activating protein encoded by the NF1 gene. Erlotinib failed to fully inhibit RAS-ERK signaling when neurofibromin levels were reduced. Treatment of neurofibromin-deficient lung cancers with a MAP-ERK kinase (MEK) inhibitor restored sensitivity to erlotinib. Low levels of NF1 expression were associated with primary and acquired resistance of lung adenocarcinomas to EGFR TKIs in patients. These findings identify a subgroup of patients with EGFR-mutant lung adenocarcinoma who might benefit from combination therapy with EGFR and MEK inhibitors.

HTS-DB: an online resource to publish and query data from functional genomics high-throughput siRNA screening projects.

High-throughput screening (HTS) uses technologies such as RNA interference to generate loss-of-function phenotypes on a genomic scale. As these technologies become more popular, many research institutes have established core facilities of expertise to deal with the challenges of large-scale HTS experiments. As the efforts of core facility screening projects come to fruition, focus has shifted towards managing the results of these experiments and making them available in a useful format that can be further mined for phenotypic discovery. The HTS-DB database provides a public view of data from screening projects undertaken by the HTS core facility at the CRUK London Research Institute. All projects and screens are described with comprehensive assay protocols, and datasets are provided with complete descriptions of analysis techniques. This format allows users to browse and search data from large-scale studies in an informative and intuitive way. It also provides a repository for additional measurements obtained from screens that were not the focus of the project, such as cell viability, and groups these data so that it can provide a gene-centric summary across several different cell lines and conditions. All datasets from our screens that can be made available can be viewed interactively and mined for further hit lists. We believe that in this format, the database provides researchers with rapid access to results of large-scale experiments that might facilitate their understanding of genes/compounds identified in their own research. DATABASE URL: http://hts.cancerresearchuk.org/db/public.

Salt-inducible kinases regulate growth through the Hippo signalling pathway in Drosophila.

The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell-cycle exit and apoptosis, is a major determinant of size control. The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). We performed a split-TEV-based genome-wide RNAi screen for modulators of Hpo signalling. We characterize the Drosophila salt-inducible kinases (Sik2 and Sik3) as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser 413. As Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hpo pathway and systemic growth control.

Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target.

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome-wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta-analysis of five ccRCC expression datasets. MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra-tumoural heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs. MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse-free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC.

Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC.

Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP-LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation.

Structural analysis of eight novel and 112 previously reported missense mutations in the interactive FXI mutation database reveals new insight on FXI deficiency.

Factor XI (FXI) functions in blood coagulation. FXI is composed of four apple (Ap) domains and a serine protease (SP) domain. Deficiency of FXI leads to an injury-related bleeding disorder, which is remarkable for the lack of correlation between bleeding symptoms and FXI coagulant activity (FXI:C). The number of mutations previously reported in our interactive web database (http://www.FactorXI.org) is now significantly increased to 183 through our new patient studies and from literature surveys. Eight novel missense mutations give a total of 120 throughout the FXI gene (F11). The most abundant defects in FXI are revealed to be those from low-protein plasma levels (Type I: CRM-) that originate from protein misfolding, rather than from functional defects (Type II: CRM+). A total of 70 Ap missense mutations were analysed using a consensus Ap domain structure generated from the FXI dimer crystal structure. This showed that all parts of the Ap domain were affected. The 47 SP missense mutations were also distributed throughout the SP domain structure. The periphery of the Ap beta-sheet structure is sensitive to structural perturbation caused by residue changes throughout the Ap domain, yet this beta-sheet is crucial for FXI dimer formation. Residues located at the Ap4:Ap4 interface in the dimer are much less directly involved. We conclude that the abundance of Type I defects in FXI results from the sensitivity of the Ap domain folding to residue changes within this, and discuss how structural knowledge of the mutations improves our understanding of FXI deficiencies.

Charge environments around phosphorylation sites in proteins.

BACKGROUND: Phosphorylation is a central feature in many biological processes. Structural analyses have identified the importance of charge-charge interactions, for example mediating phosphorylation-driven allosteric change and protein binding to phosphopeptides. Here, we examine computationally the prevalence of charge stabilisation around phosphorylated sites in the structural database, through comparison with locations that are not phosphorylated in the same structures. RESULTS: A significant fraction of phosphorylated sites appear to be electrostatically stabilised, largely through interaction with sidechains. Some examples of stabilisation across a subunit interface are evident from calculations with biological units. When considering the immediately surrounding environment, in many cases favourable interactions are only apparent after conformational change that accompanies phosphorylation. A simple calculation of potential interactions at longer-range, applied to non-phosphorylated structures, recovers the separation exhibited by phosphorylated structures. In a study of sites in the Phospho.ELM dataset, for which structural annotation is provided by non-phosphorylated proteins, there is little separation of the known phospho-acceptor sites relative to background, even using the wider interaction radius. However, there are differences in the distributions of patch polarity for acceptor and background sites in the Phospho.ELM dataset. CONCLUSION: In this study, an easy to implement procedure is developed that could contribute to the identification of phospho-acceptor sites associated with charge-charge interactions and conformational change. Since the method gives information about potential anchoring interactions subsequent to phosphorylation, it could be combined with simulations that probe conformational change. Our analysis of the Phospho.ELM dataset also shows evidence for mediation of phosphorylation effects through (i) conformational change associated with making a solvent inaccessible phospho-acceptor site accessible, and (ii) modulation of protein-protein interactions.

Association of a factor H mutation with hemolytic uremic syndrome following a diarrheal illness.

Hemolytic uremic syndrome (HUS) takes 2 forms: diarrheal HUS and nondiarrheal HUS. As its name suggests, diarrheal HUS classically follows an enteric infection. The classic infective organism is the Escherichia coli O157 serotype, although other bacteria, including Shigella species, can produce the verocytotoxin required to cause HUS. The usual clinical course is an episode of bloody diarrhea followed by thrombotic microangiopathy and acute renal failure. Supportive treatment sees recovery of renal function in the vast majority of patients. Most cases occur in children, but all age groups can be affected. Conversely, nondiarrheal HUS may have one of a number of predisposing factors, including drugs, irradiation, and hypertension. It also is well established that mutations in the genes encoding the complement regulator proteins factor H, factor I, and membrane cofactor protein predispose to nondiarrheal HUS. In patients with nondiarrheal HUS, recovery of renal function is much less common. Here, we present a case of HUS after a diarrheal illness in which the patient did not recover renal function in the long term. A novel mutation in exon 23 of the factor H gene was discovered. This is clinically important. If this patient underwent transplantation, he would be expected to have an 80% risk of graft loss at 2 years because of recurrent HUS. We recommend consideration of complement gene mutations in any patient with HUS after a diarrheal episode in which there are unusual features.

CoagMDB: a database analysis of missense mutations within four conserved domains in five vitamin K-dependent coagulation serine proteases using a text-mining tool.

Central repositories of mutations that combine structural, sequence, and phenotypic information in related proteins will facilitate the diagnosis and molecular understanding of diseases associated with them. Coagulation involves the sequential activation of serine proteases and regulators in order to yield stable blood clots while maintaining hemostasis. Five coagulation serine proteases-factor VII (F7), factor IX (F9), factor X (F10), protein C (PROC), and thrombin (F2)-exhibit high sequence similarities and all require vitamin K. All five of these were incorporated into an interactive database of mutations named CoagMDB (http://www.coagMDB.org; last accessed: 9 August 2007). The large number of mutations involved (especially for factor IX) and the increasing problem of out-of-date databases required the development of new database management tools. A text mining tool automatically scans full-length references to identify and extract mutations. High recall rates between 96 and 99% and precision rates of 87 to 93% were achieved. Text mining significantly reduces the time and expertise required to maintain the databases and offers a solution to the problem of locus-specific database management and upkeep. A total of 875 mutations were extracted from 1,279 literature sources. Of these, 116 correspond to Gla domains, 86 to the N-terminal EGF domain, 73 to the C-terminal EGF domain, and 477 to the serine protease domain. The combination of text mining and consensus domain structures enables mutations to be correlated with experimentally-measurable phenotypes based on either low protein levels (Type I) or reduced functional activities (Type II), respectively. A tendency for the conservation of phenotype with structural location was identified.

The interactive Factor H-atypical hemolytic uremic syndrome mutation database and website: update and integration of membrane cofactor protein and Factor I mutations with structural models.

Atypical hemolytic uremic syndrome (aHUS) is a disease of hemolytic anemia, thrombocytopenia, and renal failure associated with defective alternative pathway (AP) complement control. Previously, we presented a database (www.FH-HUS.org) focusing on aHUS mutations in the Factor H gene (CFH). Here, new aHUS mutations are reported for the complement regulatory proteins Factor H (FH), Factor I (FI), and membrane cofactor protein (MCP). Additional mutations or polymorphisms within CFH have been associated with membranoproliferative glomerulonephritis (MPGN) and age-related macular degeneration (AMD). Accordingly, the database now includes substitutions that predispose to aHUS, MPGN, and AMD. For this, structural models for the domains in MCP and FI were developed using homology modeling. With this new database, patients with mutations in more than one gene can be displayed and interpreted in a coherent manner. The database also includes SNP polymorphisms in CFH, MCP, and IF. There are now a total of 167 genetic alterations, including 100 in CFH, 43 in MCP, and 24 in IF. The mutations characterize clinical outcomes that vary from several AMD-associated polymorphisms to those associated with aHUS, MPGN, or FI deficiency. A consensus short complement regulator (SCR) domain structure facilitated the interpretations of aHUS mutations. Specific locations within this consensus domain often correlate with the occurrence of clinical phenotypes. The AMD Tyr402His polymorphism is structurally located at a hotspot for several aHUS mutations. The database emphasizes the causative role of the alternative pathway of complement in disease and provides a repository of knowledge to assist future diagnosis and novel therapeutic approaches.

A user's guide to the interactive Web database of factor H-associated hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) mutations have been reported in the complement regulatory proteins factor H, factor I, and membrane cofactor protein (MCP). Mutations within factor H are also associated with membranoproliferative glomerulonephritis and age-related macular degeneration. The increasing amount of information on aHUS requires organization if it is to be usable. Accordingly, an interactive factor H aHUS Web database has been developed (http://www.fh-hus.org) that integrates genotypic, phenotypic, and structural information for mutations within human factor H. This provides a valuable tool for the interpretation of previously reported aHUS mutations, and provides prediction and analysis tools for new mutations. It will be extended to include mutations in factor I and MCP. Here, we describe how to use this Web database as a research tool, and indicate possible future directions depending on feedback from the clinical community.

An interactive web database of factor H-associated hemolytic uremic syndrome mutations: insights into the structural consequences of disease-associated mutations.

Factor H (FH) is a central complement regulator comprised of 20 short complement repeat (SCR) domains. Nucleotide changes within this gene (CFH) have been observed in patients with hemolytic uremic syndrome (HUS), and also membranoproliferative glomerulonephritis and age-related macular degeneration. All parts of FH are affected, but many mutations are clustered in the C-terminal part of FH. Up to now, structural analyses of HUS have been based on SCR-20, a domain that is involved in FH interactions with C3b, heparin, and endothelial cells. In order to identify the structural and functional consequence of HUS mutations, further disease-associated mutations were analyzed in terms of homology and nuclear magnetic resonance (NMR) models for factor H SCR domains. An interactive web database of 54 human HUS-associated mutations and others was created from the literature (www.FH-HUS.org). This has comprehensive search and analysis tools, integrating phenotypic and genetic data with structural analysis. Each mutation can be highlighted on the SCR structure together with the patient FH and C3 levels where available. Two new insights were obtained from our collection of data. First, phenotypic data on FH clarify our previously-proposed classification of Type I and Type II disorders that both lead to HUS, where Type I affects FH secretion and folding, and Type II leads to expressed protein in plasma that is functionally defective. Second, the new mutations show more clearly that SCR domains from SCR-16 to SCR-19 are important for the ligand binding activities of FH as well as SCR-20. This FH web database will facilitate the interpretation of new mutations and polymorphisms when these are identified in patients, and it will clarify the functional role of FH.

Factor H and atypical hemolytic uremic syndrome: mutations in the C-terminus cause structural changes and defective recognition functions.

Atypical hemolytic uremic syndrome is a disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations in the complement regulator factor H are associated with the inherited form of the disease, and >60% of the mutations are located within the C terminus of factor H. The C-terminus of factor H, represented by short consensus repeat 19 (SCR19) and SCR20, harbors multiple functions; consequently, this study aimed to examine the functional effects of clinically reported mutations in these SCR. Mutant factor H proteins (W1157R, W1183L, V1197A, R1210C, R1215G, and P1226S) were recombinantly expressed and functionally characterized. All six mutant proteins showed severely reduced heparin, C3b, C3d, and endothelial cell binding. By peptide spot analyses, four linear regions that are involved in heparin, C3b, and C3d binding were localized in SCR19 and SCR20. A three-dimensional homology model of the two domains suggests that these four regions form a common binding site across both domains. In addition, this structural model identifies two types of residues: Type A residues are positioned on the SCR surface and are represented by mutants W1157R, W1183L, R1210C, and R1215G; and type B residues are buried within the SCR structure and affect mutations V1197A and P1226S. Mutations of both types of residue result in the same functional defects, namely the reduced binding of factor H to surface-attached C3b molecules and reduced complement regulatory activity at the cell surfaces. The buried type B mutations seem to affect ligand interaction of factor H more severely than the surface-exposed mutations.