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Anastassis Perrakis

Publications and source records attributed to Anastassis Perrakis.

18 recordsLinked to original sources
PubMed2025

Zincore, an atypical coregulator, binds zinc finger transcription factors to control gene expression.

Zinc finger proteins (ZNFs) are the largest family of transcription factors, yet how they activate gene expression remains unclear. In this study, we identified Zincore, a protein complex consisting of QRICH1 and SEPHS1, as a ZNF-specific coregulator essential for embryonic development in mice and associated with developmental syndromes in humans. We also identified ZFP91 as a representative Zincore client, binding the conserved promoter motif CTTTAAR. Cryo-electron microscopy of a Zincore-ZFP91-DNA complex revealed a SEPHS1 arginine clamp to recognize the DNA-bound zinc finger domains. This mode of binding explains recognition of different ZNFs and stabilizes ZFP91 onto its cognate DNA motif. Thus, our study identified Zincore as a ZNF-specific coregulator essential for development, involving a distinctive mechanism that locks ZNFs onto DNA and regulates transcription.

Animals↗
PubMed2006

Assessment of automatic ligand building in ARP/wARP.

The efficiency of the ligand-building module of ARP/wARP version 6.1 has been assessed through extensive tests on a large variety of protein-ligand complexes from the PDB, as available from the Uppsala Electron Density Server. Ligand building in ARP/wARP involves two main steps: automatic identification of the location of the ligand and the actual construction of its atomic model. The first step is most successful for large ligands. The second step, ligand construction, is more powerful with X-ray data at high resolution and ligands of small to medium size. Both steps are successful for ligands with low to moderate atomic displacement parameters. The results highlight the strengths and weaknesses of both the method of ligand building and the large-scale validation procedure and help to identify means of further improvement.

Algorithms↗
PubMed2006

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.

Algorithms↗
PubMed2006

Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery.

DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b.

Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.

Amino Acid Sequence↗
PubMed2005

Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG+ strategy.

A crystallization screening process is presented that was developed for a small academic laboratory. Its underlying concept is to combine sparse-matrix screening with systematic screening in a minimum number of crystallization conditions. The sparse-matrix screen is the cherry-picked combination of conditions from the Joint Center for Structural Genomics (JCSG) extended using conditions from other screens. Its aim is to maximize the coverage of crystallization parameter space with no redundancy. The systematic screen, a pH-, anion- and cation-testing (PACT) screen, aims to decouple the components of each condition and to provide information about the protein, even in the absence of crystals, rather than cover a wide crystallization space. This screening strategy is combined with nanolitre-volume dispensing hardware and a small but practical experiment-tracking system. The screens have been tested both at the NKI and in other laboratories and it is concluded that they provide a useful minimal screening strategy.

Algorithms↗
PubMed2005

Modeling and experimental validation of the binary complex of the plectin actin-binding domain and the first pair of fibronectin type III (FNIII) domains of the beta4 integrin.

The binding of plectin to the beta4 subunit of the alpha6beta4 integrin is a critical step in the formation of hemidesmosomes. An important interaction between these two proteins occurs between the actin-binding domain (ABD) of plectin and the first pair of fibronectin type III (FNIII) domains and a small part of the connecting segment of beta4. Previously, a few amino acids, critical for this interaction, were identified in both plectin and beta4 and mapped on the crystal structures of the ABD of plectin and the first pair of FNIII domains of beta4. In the present study, we used this biochemical information and protein-protein docking calculations to construct a model of the binary complex between these two protein domains. The top scoring computational model predicts that the calponin-homology 1 (CH1) domain of the ABD associates with the first and the second FNIII domains of beta4. Our mutational analysis of the residues at the proposed interface of both the FNIII and the CH1 domains is in agreement with the suggested interaction model. Computational simulations to predict protein motions suggest that the exact model of FNIII and plectin CH1 interaction might well differ in detail from the suggested model due to the conformational plasticity of the FNIII domains, which might lead to a closely related but different mode of interaction with the plectin-ABD. Furthermore, we show that Ser-1325 in the connecting segment of beta4 appears to be essential for the recruitment of plectin into hemidesmosomes in vivo. This is consistent with the proposed model and previously published mutational data. In conclusion, our data support a model in which the CH1 domain of the plectin-ABD associates with the groove between the two FNIII domains of beta4.

Actins↗
PubMed2005

Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate.

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Allosteric Site↗
PubMed2004

Developments in the CCP4 molecular-graphics project.

Progress towards structure determination that is both high-throughput and high-value is dependent on the development of integrated and automatic tools for electron-density map interpretation and for the analysis of the resulting atomic models. Advances in map-interpretation algorithms are extending the resolution regime in which fully automatic tools can work reliably, but at present human intervention is required to interpret poor regions of macromolecular electron density, particularly where crystallographic data is only available to modest resolution [for example, I/sigma(I) < 2.0 for minimum resolution 2.5 A]. In such cases, a set of manual and semi-manual model-building molecular-graphics tools is needed. At the same time, converting the knowledge encapsulated in a molecular structure into understanding is dependent upon visualization tools, which must be able to communicate that understanding to others by means of both static and dynamic representations. CCP4 mg is a program designed to meet these needs in a way that is closely integrated with the ongoing development of CCP4 as a program suite suitable for both low- and high-intervention computational structural biology. As well as providing a carefully designed user interface to advanced algorithms of model building and analysis, CCP4 mg is intended to present a graphical toolkit to developers of novel algorithms in these fields.

Algorithms↗
PubMed2004

Towards complete validated models in the next generation of ARP/wARP.

The design of a new versatile control system that will underlie future releases of the automated model-building package ARP/wARP is presented. A sophisticated expert system is under development that will transform ARP/wARP from a very useful model-building aid to a truly automated package capable of delivering complete, well refined and validated models comparable in quality to the result of intensive manual checking, rebuilding, hypothesis testing, refinement and validation cycles of an experienced crystallographer. In addition to the presentation of this control system, recent advances, ideas and future plans for improving the current model-building algorithms, especially for completing partially built models, are presented. Furthermore, a concept for integrating validation routines into the iterative model-building process is also presented.

Algorithms↗
PubMed2004

Crystal structure of the targeting endonuclease of the human LINE-1 retrotransposon.

The human L1 endonuclease (L1-EN) is encoded by the non-LTR retrotransposon LINE-1 (L1). L1 is responsible for more than 1.5 million retrotransposition events in the history of the human genome, contributing more than a quarter to human genomic DNA (L1 and Alu elements). L1-EN is related to the well-understood human DNA repair endonuclease APE1, and its nicking specificity is a major determinant for retrotransposon integration site selection. The crystal structure of human L1 endonuclease is the first of a retrotransposon-encoded protein and a prototype for retrotransposon-encoded endonucleases involved in target-primed reverse transcription. Structure-based endonuclease alignments reveal a conserved threonine in addition to previously identified invariant residues and suggest that DNA recognition proceeds via the accommodation of an extrahelical nucleotide within a pocket of the enzyme. The present analysis will help to refine phylogenetic and functional relationships among metal-dependent phosphohydrolases and provides a basis for manipulating non-LTR retrotransposon integration site selection.

Amino Acid Sequence↗
PubMed2003

Breaking good resolutions with ARP/wARP.

New procedures are outlined that enable ARP/wARP to automatically build protein models with diffraction data extending to about 2.5 A. An overview of ongoing research is given and possible future advances are discussed.

Algorithms↗
PubMed2003

Structures of Escherichia coli DNA mismatch repair enzyme MutS in complex with different mismatches: a common recognition mode for diverse substrates.

We have refined a series of isomorphous crystal structures of the Escherichia coli DNA mismatch repair enzyme MutS in complex with G:T, A:A, C:A and G:G mismatches and also with a single unpaired thymidine. In all these structures, the DNA is kinked by approximately 60 degrees upon protein binding. Two residues widely conserved in the MutS family are involved in mismatch recognition. The phenylalanine, Phe 36, is seen stacking on one of the mismatched bases. The same base is also seen forming a hydrogen bond to the glutamate Glu 38. This hydrogen bond involves the N7 if the base stacking on Phe 36 is a purine and the N3 if it is a pyrimidine (thymine). Thus, MutS uses a common binding mode to recognize a wide range of mismatches.

Adenosine Triphosphatases↗
PubMed2003

Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation.

The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein. The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F used in our structural studies, residues 409-426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. We show by solution studies that a second site, probably contained within the "marked box" region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own. In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2cyclin DE. Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.

Blotting, Western↗
PubMed2002

Ptprj is a candidate for the mouse colon-cancer susceptibility locus Scc1 and is frequently deleted in human cancers.

Only a small proportion of cancers result from familial cancer syndromes with Mendelian inheritance. Nonfamilial, 'sporadic' cancers, which represent most cancer cases, also have a significant hereditary component, but the genes involved have low penetrance and are extremely difficult to detect. Therefore, mapping and cloning of quantitative trait loci (QTLs) for cancer susceptibility in animals could help identify homologous genes in humans. Several cancer-susceptibility QTLs have been mapped in mice and rats, but none have been cloned so far. Here we report the positional cloning of the mouse gene Scc1 (Susceptibility to colon cancer 1) and the identification of Ptprj, encoding a receptor-type protein tyrosine phosphatase, as the underlying gene. In human colon, lung and breast cancers, we show frequent deletion of PTPRJ, allelic imbalance in loss of heterozygosity (LOH) and missense mutations. Our data suggest that PTPRJ is relevant to the development of several different human cancers.

Adenocarcinoma↗
PubMed2002

ARP/wARP's model-building algorithms. I. The main chain.

Algorithms underlying the automatic model-building functionality of the ARP/wARP software suite are presented. Finding the most likely set of Calpha atoms from a given set of atoms is formulated as a constrained integer programming problem. The objective function is a density-weighted score for the match between observed and expected chain conformation. Graph-search algorithms are presented that find solutions to this problem in an efficient manner.

Algorithms↗