Plastin 3 in health and disease: a matter of balance.

For a long time, PLS3 (plastin 3, also known as T-plastin or fimbrin) has been considered a rather inconspicuous protein, involved in F-actin-binding and -bundling. However, in recent years, a plethora of discoveries have turned PLS3 into a highly interesting protein involved in many cellular processes, signaling pathways, and diseases. PLS3 is localized on the X-chromosome, but shows sex-specific, inter-individual and tissue-specific expression variability pointing towards skewed X-inactivation. PLS3 is expressed in all solid tissues but usually not in hematopoietic cells. When escaping X-inactivation, PLS3 triggers a plethora of different types of cancers. Elevated PLS3 levels are considered a prognostic biomarker for cancer and refractory response to therapies. When it is knocked out or mutated in humans and mice, it causes osteoporosis with bone fractures; it is the only protein involved in actin dynamics responsible for osteoporosis. Instead, when PLS3 is upregulated, it acts as a highly protective SMN-independent modifier in spinal muscular atrophy (SMA). Here, it seems to counteract reduced F-actin levels by restoring impaired endocytosis and disturbed calcium homeostasis caused by reduced SMN levels. In contrast, an upregulation of PLS3 on wild-type level might cause osteoarthritis. This emphasizes that the amount of PLS3 in our cells must be precisely balanced; both too much and too little can be detrimental. Actin-dynamics, regulated by PLS3 among others, are crucial in a lot of cellular processes including endocytosis, cell migration, axonal growth, neurotransmission, translation, and others. Also, PLS3 levels influence the infection with different bacteria, mycosis, and other pathogens.

Expression and Localization of Thrombospondins, Plastin 3, and STIM1 in Different Cartilage Compartments of the Osteoarthritic Varus Knee.

Osteoarthritis (OA) is a multifactorial disease which is characterized by a change in the homeostasis of the extracellular matrix (ECM). The ECM is essential for the function of the articular cartilage and plays an important role in cartilage mechanotransduction. To provide a better understanding of the interaction between the ECM and the actin cytoskeleton, we investigated the localization and expression of the Ca2+-dependent proteins cartilage oligomeric matrix protein (COMP), thrombospondin-1 (TSP-1), plastin 3 (PLS3) and stromal interaction molecule 1 (STIM1). We investigated 16 patients who suffered from varus knee OA and performed a topographical analysis of the cartilage from the medial and lateral compartment of the proximal tibial plateau. In a varus knee, OA is more pronounced in the medial compared to the lateral compartment as a result of an overloading due to the malalignment. We detected a location-dependent staining of PLS3 and STIM1 in the articular cartilage tissue. The staining intensity for both proteins correlated with the degree of cartilage degeneration. The staining intensity of TSP-1 was clearly reduced in the cartilage of the more affected medial compartment, an observation that was confirmed in cartilage extracts by immunoblotting. The total amount of COMP was unchanged; however, slight changes were detected in the localization of the protein. Our results provide novel information on alterations in OA cartilage suggesting that Ca2+-dependent mechanotransduction between the ECM and the actin cytoskeleton might play an essential role in the pathomechanism of OA.

Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation.

Caspase-6 is a cysteine protease implicated in neuronal survival and apoptosis. Deregulation of caspase-6 activity was linked to several neurodegenerative disorders including Alzheimer's and Huntington's Diseases. Several recent studies on the structure of caspase-6 feature the caspase-6 zymogen, mature apo-caspase-6 as well as the Ac-VEID-CHO peptide complex. All structures share the same typical dimeric caspase conformation. However, mature apo-caspase-6 crystallized at low pH revealed a novel, non-canonical inactive caspase conformation speculated to represent a latent state of the enzyme suitable for the design of allosteric inhibitors. In this treatise we present the structure of caspase-6 in the non-canonical inactive enzyme conformation bound to the irreversible inhibitor Z-VAD-FMK. The complex features a unique peptide binding mode not observed previously.

A comparative study of fragment screening methods on the p38α kinase: new methods, new insights.

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.

Biases in Drosophila melanogaster protein trap screens.

BACKGROUND: The ability to localise or follow endogenous proteins in real time in vivo is of tremendous utility for cell biology or systems biology studies. Protein trap screens utilise the random genomic insertion of a transposon-borne artificial reporter exon (e.g. encoding the green fluorescent protein, GFP) into an intron of an endogenous gene to generate a fluorescent fusion protein. Despite recent efforts aimed at achieving comprehensive coverage of the genes encoded in the Drosophila genome, the repertoire of genes that yield protein traps is still small. RESULTS: We analysed the collection of available protein trap lines in Drosophila melanogaster and identified potential biases that are likely to restrict genome coverage in protein trap screens. The protein trap screens investigated here primarily used P-element vectors and thus exhibit some of the same positional biases associated with this transposon that are evident from the comprehensive Drosophila Gene Disruption Project. We further found that protein trap target genes usually exhibit broad and persistent expression during embryonic development, which is likely to facilitate better detection. In addition, we investigated the likely influence of the GFP exon on host protein structure and found that protein trap insertions have a significant bias for exon-exon boundaries that encode disordered protein regions. 38.8% of GFP insertions land in disordered protein regions compared with only 23.4% in the case of non-trapping P-element insertions landing in coding sequence introns (p < 10(-4)). Interestingly, even in cases where protein domains are predicted, protein trap insertions frequently occur in regions encoding surface exposed areas that are likely to be functionally neutral. Considering the various biases observed, we predict that less than one third of intron-containing genes are likely to be amenable to trapping by the existing methods. CONCLUSION: Our analyses suggest that the utility of P-element vectors for protein trap screens has largely been exhausted, and that approximately 2,800 genes may still be amenable using piggyBac vectors. Thus protein trap strategies based on current approaches are unlikely to offer true genome-wide coverage. We suggest that either transposons with reduced insertion bias or recombineering-based targeting techniques will be required for comprehensive genome coverage in Drosophila.

Analysis of complex protein kinase B signalling pathways in human prostate cancer samples.

OBJECTIVE: To provide a rational basis for targeted treatment approaches in prostate cancer deregulation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) system was analysed. PATIENTS AND METHODS: In all, 45 patients with primary localized prostate cancer that underwent radical prostatectomy were included in the present study. Upon scoring of the pathological grade, expression and phosphorylation levels of PKB/Akt and relevant downstream targets were determined in tissue specimens by immunohistochemistry using specific antibodies against PTEN, PKB/Akt, its downstream targets, and the respective phosphorylated proteins. RESULTS: Most patients (>90%) had up-regulated expression and/or phosphorylation of PKB/Akt in the malignant tissue compared with the surrounding benign tissue, with a higher prevalence of increased phosphorylated PKB/Akt in patients with Gleason scores of > or =6 (100%) compared with those with Gleason scores of 4-5 (five of 13 patients), and in particular in patients with clinical progression. Up-regulated phosphorylation of PKB/Akt occurred either in association with loss or inactivation of PTEN or in a PTEN-independent manner. Enhanced phosphorylation levels of the PKB/Akt substrates glycogen synthase kinase 3, the mammalian target of rapamycin or the forkhead transcription factor like 1 (FKHRL1) were found in 29%, 42% and 40% of the tumours, respectively. Of these, only increased phosphorylated-FKHRL1 levels correlated with clinical progression. Interestingly, 80% of patients had a notable overexpression but not phosphorylation of the eucaryotic initiation factor 4E binding protein. CONCLUSION: Deregulation of p-PKB/Akt is common in localized prostate cancer and has a putative value as predictive marker for disease progression and as therapeutic target. However, as a consequence of the substantial heterogeneity in the expression and phosphorylation levels of relevant PKB/Akt effector pathways, for a rational use of specified inhibitors of the PI3K/PKB system a complex pattern testing of expression and activity of the respective target proteins for prediction of efficacy and prognosis seems mandatory.

Guidelines for the successful generation of protein-ligand complex crystals.

With continuous technical improvements at synchrotron facilities, data-collection rates have increased dramatically. This makes it possible to collect diffraction data for hundreds of protein-ligand complexes within a day, provided that a suitable crystal system is at hand. However, developing a suitable crystal system can prove challenging, exceeding the timescale of data collection by several orders of magnitude. Firstly, a useful crystallization construct of the protein of interest needs to be chosen and its expression and purification optimized, before screening for suitable crystallization and soaking conditions can start. This article reviews recent publications analysing large data sets of crystallization trials, with the aim of identifying factors that do or do not make a good crystallization construct, and gives guidance in the design of an expression construct. It provides an overview of common protein-expression systems, addresses how ligand binding can be both help and hindrance for protein purification, and describes ligand co-crystallization and soaking, with an emphasis on troubleshooting.

Array-based comparative gene expression analysis of tumor cells with increased apoptosis resistance after hypoxic selection.

Tumor hypoxia is an adverse prognostic factor. In a recent study, we could demonstrate that cyclic hypoxia selects for hypoxia-tolerant tumor cells, which are cross-resistant to other stimuli of mitochondrial death pathways. In contrast, sensitivity of the cells to death-receptor ligands was mainly not affected. The aim of the present study was to further elucidate cellular changes induced by cyclic hypoxia and to identify alterations in gene expression pattern upon hypoxic selection by means of DNA-microarray analysis. Our data reveal that cyclic hypoxia resulted in the selection of cells with resistance to doxorubicine and radiation. Furthermore, hypoxic selection was accompanied by constitutive changes of the gene expression pattern with downregulation of 156 and upregulation of 82 genes. Most of the differentially regulated genes were involved in cellular responses to hypoxia and reoxygenation. While many of the genes that were downregulated upon hypoxic selection represent genes that are usually upregulated by acute hypoxia, the genes that were upregulated represent genes that are involved in stress resistance and anti-apoptotic signalling. Most importantly, hypoxic selection was not associated with changes of single apoptosis relevant genes, but with alterations in gene expression levels of a wide variety of genes indicating a more complex adaptation process.

Identification and Investigation of Novel Binding Fragments in the Fatty Acid Binding Protein 6 (FABP6).

Fatty acid binding protein 6 (FABP6) is a potential drug discovery target, which, if inhibited, may have a therapeutic benefit for the treatment of diabetes. Currently, there are no published inhibitors of FABP6, and with the target believed to be amenable to fragment-based drug discovery, a structurally enabled program was initiated. This program successfully identified fragment hits using the surface plasmon resonance (SPR) platform. Several hits were validated with SAR and were found to be displaced by the natural ligand taurocholate. We report the first crystal structure of human FABP6 in the unbound form, in complex with cholate, and with one of the key fragments.

Intracellular mediators of erucylphosphocholine-induced apoptosis.

Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNFalpha-related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c, activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway.

Structure and biosynthesis of cetoniacytone A, a cytotoxic aminocarba sugar produced by an endosymbiontic Actinomyces.

Cetoniacytone A (1) and some related minor components (2, 6, 7) were produced by Actinomyces sp. (strain Lu 9419), which was isolated from the intestines of a rose chafer (Cetonia aureata). The structures of the novel metabolites were established by detailed spectroscopic analysis. The absolute configuration of 1 was determined by X-ray analysis and derivatisation with chiral acids. 1 exhibits a significant cytotoxicity against selected tumor cell lines. The biosynthesis of 1 was studied by feeding 13C labelled precursors. The results suggest that the characteristic p-C7N skeleton of the aminocarba sugar is formed via the pentose phosphate pathway by cyclisation of a heptulose phosphate intermediate.

Design, synthesis, and biological evaluation of potent and selective class IIa histone deacetylase (HDAC) inhibitors as a potential therapy for Huntington's disease.

Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntington's disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a "closed-loop" form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.

A new apo-caspase-6 crystal form reveals the active conformation of the apoenzyme.

Caspase-6 has been identified as a key component in the pathway of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. It has been the focus of drug development for some time, but only recently have structural data become available. The first study identified a novel noncanonical conformation of apo-caspase-6 contrasting with the typical caspase conformation. Then, the structures of both caspase-6 zymogen and the Ac-VEID-CHO peptide inhibitor complex described caspase-6 in the canonical conformation, raising the question of why the intermediate between these two structures (mature apo-caspase-6) would adopt the noncanonical conformation. In this study, we present a new crystal form of the apoenzyme in the canonical conformation by identifying the previous apostructure as a pH-inactivated form of caspase-6. Our new apostructure is further compared to the Ac-VEID-CHO caspase-6 inhibitor complex. The structural comparison allows us to visualize the organization of loops L2, L3, and L4 upon ligand binding and how the catalytic groove forms to accommodate the inhibitor.

Succinate complex crystal structures of the alpha-ketoglutarate-dependent dioxygenase AtsK: steric aspects of enzyme self-hydroxylation.

The alkylsulfatase AtsK from Pseudomonas putida S-313 is a member of the non-heme iron(II)-alpha-ketoglutarate-dependent dioxygenase superfamily. In the initial step of their catalytic cycle, enzymes belonging to this widespread and versatile family coordinate molecular oxygen to the iron center in the active site. The subsequent decarboxylation of the cosubstrate alpha-ketoglutarate yields carbon dioxide, succinate, and a highly reactive ferryl (IV) species, which is required for substrate oxidation via a complex mechanism involving the transfer of radical species. Non-productive activation of oxygen may lead to harmful side reactions; therefore, such enzymes need an effective built-in protection mechanism. One of the ways of controlling undesired side reactions is the self-hydroxylation of an aromatic side chain, which leads to an irreversibly inactivated species. Here we describe the crystal structure of the alkylsulfatase AtsK in complexes with succinate and with Fe(II)/succinate. In the crystal structure of the AtsK-Fe(II)-succinate complex, the side chain of Tyr(168) is co-ordinated to the iron, suggesting that Tyr(168) is the target of enzyme self-hydroxylation. This is the first structural study of an Fe(II)-alpha-ketoglutarate-dependent dioxygenase that presents an aromatic side chain coordinated to the metal center, thus allowing structural insight into this protective mechanism of enzyme self-inactivation.

Apoptotic response of Chang cells to infection with Pseudomonas aeruginosa strains PAK and PAO-I: molecular ordering of the apoptosis signaling cascade and role of type IV pili.

Pseudomonas aeruginosa is a gram-negative facultative opportunistic pathogen associated with severe infections in immunocompromised hosts and in patients with cystic fibrosis. P. aeruginosa strains show divergent pathogenicity in vivo and trigger apoptosis of and/or are internalized into human host cells. In the present study, we studied the molecular ordering of apoptosis signaling upon infection of human conjunctiva epithelial Chang cells with P. aeruginosa PAK as well as the role of bacterial pili in the response to the infection. Our results show that CD95 up-regulation is followed by early activation of caspase-8 and -3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase. The data also demonstrate release of apoptosis inducing factor into the cytosol of infected cells. Induction of mitochondrial alterations, i.e., mitochondrial depolarization and release of cytochrome c, as well as cleavage of caspase-9, -7, and -1 occurred only at later time points. In addition, our results demonstrate that pili are required for P. aeruginosa-induced apoptosis of human epithelial cells. While the two piliated P. aeruginosa strains, PAO-I and PAK, induced apoptosis of Chang cells within 3 h of infection, the pilus-deficient P. aeruginosa mutants PAK Delta pilA and PAK Delta pilA Delta all were without effect. The pilus-deficient mutants failed to induce a significant up-regulation of CD95 on the cell surface and to trigger mitochondrial alterations or activation of caspase-8, -3, and -7. In addition, only the piliated wild-type strains induced caspase-1-mediated activation of interleukin-1 beta. Thus, pili are necessary for distinct infection-induced cellular responses of human epithelial cells.

Crystal structure of the alkylsulfatase AtsK: insights into the catalytic mechanism of the Fe(II) alpha-ketoglutarate-dependent dioxygenase superfamily.

The alkylsulfatase AtsK from Pseudomonas putida S-313 belongs to the widespread and versatile non-heme iron(II) alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes the oxygenolytic cleavage of a variety of different alkyl sulfate esters to the corresponding aldehyde and sulfate. The enzyme is only expressed under sulfur starvation conditions, providing a selective advantage for bacterial growth in soils and rhizosphere. Here we describe the crystal structure of AtsK in the apo form and in three complexes: with the cosubstrate alpha-ketoglutarate, with alpha-ketoglutarate and iron, and finally with alpha-ketoglutarate, iron, and an alkyl sulfate ester used as substrate in catalytic studies. The overall fold of the enzyme is closely related to that of the taurine/alpha-ketoglutarate dioxygenase TauD and is similar to the fold observed for other members of the enzyme superfamily. From comparison of these structures with the crystal structure of AtsK and its complexes, we propose a general mechanism for the catalytic cycle of the alpha-ketoglutarate-dependent dioxygenase superfamily.