Building and assessing atomic models of proteins from structural templates: Learning and benchmarks

One approach to predict a protein fold from a sequence (a target) is based on structures of related proteins that are used as templates. We present an algorithm that examines a set of candidates for templates, builds from each of the templates an atomically detailed model, and ranks the models. The algorithm performs a hierarchical selection of the best model using a diverse set of signals. After a quick and suboptimal screening of template candidates from the protein data bank, the current method fine‐tunes the selection to a few models. More detailed signals test the compatibility of the sequence and the proposed structures, and are merged to give a global fitness measure using linear programming. This algorithm is a component of the prediction server LOOPP ( http://www.loopp.org ). Large‐scale training and tests sets were designed and are presented. Recent results of the LOOPP server in CASP8 are discussed. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Quinuclidinone O-Alkynyloximes with muscarinic agonist activity.

A series of quinuclidinone O-alkynyloximes (14-19) were synthesized and evaluated in radioligand displacement assays for binding affinities to M1-M3 muscarinic receptors. Radioligand displacement assays were carried out using [3H] oxotremorine-M and [3H] pirenzepine on rat cortical tissue and [3H] N-methylscopolamine on rat heart and submandibulary glands. Two alkynyloximes 15 and 18 had pirenzepine/oxotremorine M ratios which were indicative of muscarinic agonist and partial agonist activity, respectively. They were tested for their mnemonic effects in mice using the swimming escape task and found to attenuate scopolamine induced impairment of the task in mice at 2mg/kg. The results show that the O-alkynyloxime moiety linked to azacycles of appropriate size and rigidity (for example quinuclidine and tropane) is a potentially useful muscarinic pharmacophore that can be exploited for the design of muscarinic agonists.     

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347

NEMO is an essential regulatory component of the IκB kinase (IKK) complex, which controls activation of the NF-κB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H₂O₂) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H₂O₂ decreases TNFα-induced IKK activity in NEMO-reconstituted cells, and that TNFα has a diminished ability to activate NF-κB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.     

Microtubule disruption inhibits autophagosome-lysosome fusion: implications for studying the roles of aggresomes in polyglutamine diseases

Large cytoplasmic inclusions called aggresomes are seen in many protein conformational diseases including Huntington’s disease and Parkinson’s disease. The roles of inclusions and aggresomes in these diseases are unresolved critical issues that have been vigorously debated. Two recent studies used microtubule disruption with nocodazole to inhibit aggresome formation and observed increased toxicity of expanded polyglutamines in the context of huntingtin exon 1 and a truncated androgen receptor. Increased toxicity of expanded polyglutamines in the presence of nocodazole was correlated with decreased protein turnover, leading the authors to conclude that aggresomes were cytoprotective and that they directly enhanced clearance of the toxic proteins. Here we show that nocodazole has additional effects, which provide a simple alternative explanation for these previous observations. We confirmed aggresome formation in cells expressing proteins with polyalanine and polyglutamine expansions. As expected, we found a reduction in aggresome formation when microtubule function was disrupted using nocodazole. However, in addition to this effect, nocodazole treatment increased the proportions of cells with nuclear inclusions in PC12 cells expressing huntingtin exon 1 with 74 glutamines. This can be explained as nocodazole inhibits autophagosome-lysosome fusion, a key step in mutant huntingtin exon 1 clearance. This effect alone can explain the previous observations with this compound in polyglutamine diseases and raises doubts about the interpretation of some of the data that have been used to argue that aggresomes protect against polyglutamine mutations.     

Alpha-Synuclein is degraded by both autophagy and the proteasome

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of aggregates (Lewy bodies) in neurons. alpha-Synuclein is the major protein in Lewy bodies and rare mutations in alpha-synuclein cause early-onset PD. Consequently, alpha-synuclein is implicated in the pathogenesis of PD. Here, we have investigated the degradation pathways of alpha-synuclein, using a stable inducible PC12 cell model, where the expression of exogenous human wild-type, A30P, or A53T alpha-synuclein can be switched on and off. We have used a panel of inhibitors/stimulators of autophagy and proteasome function and followed alpha-synuclein degradation in these cells. We found that not only is alpha-synuclein degraded by the proteasome, but it is also degraded by autophagy. A role for autophagy was further supported by the presence of alpha-synuclein in organelles with the ultrastructural features of autophagic vesicles. Since rapamycin, a stimulator of autophagy, increased clearance of alpha-synuclein, it merits consideration as a potential therapeutic for Parkinsons disease, as it is designed for chronic use in humans.     

Autophagy and its possible roles in nervous system diseases, damage and repair

Increased numbers of autophagosomes/autophagic vacuoles are seen in a variety of physiological and pathological states in the nervous system. In many cases, it is unclear if this phenomenon is the result of increased autophagic activity or decreased autophagosome-lysosome fusion. The functional significance of autophagy and its relationship to cell death in the nervous system is also poorly understood. In this review, we have considered these issues in the context of acute neuronal injury and a range of chronic neurodegenerative conditions, including the Lurcher mouse, Alzheimer's, Parkinson's, Huntington's and prion diseases. While many issues remain unresolved, these conditions raise the possibility that autophagy can have either deleterious or protective effects depending on the specific situation and stage in the pathological process.     

The rocky road to personalized medicine in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a malignant disorder of the myeloid blood lineage characterized by impaired differentiation and increased proliferation of hematopoietic precursor cells. Recent technological advances have led to an improved understanding of AML biology but also uncovered the enormous cytogenetic and molecular heterogeneity of the disease. Despite this heterogeneity, AML is mostly managed by a ‘one‐size‐fits‐all’ approach consisting of intensive, highly toxic induction and consolidation chemotherapy. These treatment protocols have remained largely unchanged for the past several decades and only lead to a cure in approximately 30–35% of cases. The advent of targeted therapies in chronic myeloid leukaemia and other malignancies has sparked hope to improve patient outcome in AML. However, the implementation of targeted agents in AML therapy has been unexpectedly cumbersome and remains a difficult task due to a variety of disease‐ and patient‐specific factors. In this review, we describe current standard and investigational therapeutic strategies with a focus on targeted agents and highlight potential tools that might facilitate the development of targeted therapies for this fatal disease. The classes of agents described in this review include constitutively activated signalling pathway inhibitors, surface receptor targets, epigenetic modifiers, drugs targeting the interaction of the hematopoietic progenitor cell with the stroma and drugs that target the apoptotic machinery. The clinical context and outcome with these agents will be examined to gain insight about their optimal utilization.     

A quantitative analysis of excluded-site effects for highly cooperative binding systems

Binding isotherms can provide quantitive information regarding the stability of a molecular complex. Theorectical studies in recent years have been directed to systems in which a single ligand can exclude more than one polymer site (excluded-site effect). This system has minium of thre parameters to describe the binding data: the intrinsic binding constant, B; the remote-neighbor cooperative paramaters, σ_q and the number of excluded sites, q. It is suggested in the present communication that precise values for these three parameters can be obtained by utilizing the characteristics of two forms of data representation: θ vs ln m and θ/m vs θ, where θ is the degree of saturation (0?θ?1) and m is the molality of free ligand. The matrix generation method is used to obtain empirical equations relating the midpoint location and slope at the midpoint of the θ vs ln m plot to the three molecular parameters. A modified Scatchard theory is also presented for highly cooperative systems, which results in an expression relaing the maximum in the θ/m vs θ plot to the molecular parameters σ_q and q, thus providing the third equation for the three unknown parameters. The novel method f analysis is illustrated with the AMP-poly(L -arginine) and oligocytidylate–T7 DNA sstems.     

Contour length and refolding rate of a small protein controlled by engineered disulfide bonds

The introduction of disulfide bonds into proteins creates additional mechanical barriers and limits the unfolded contour length (i.e., the maximal extension) measured by single-molecule force spectroscopy. Here, we engineer single disulfide bonds into four different locations of the human cardiac titin module (I27) to control the contour length while keeping the distance to the transition state unchanged. This enables the study of several biologically important parameters. First, we are able to precisely determine the end-to-end length of the transition state before unfolding (53 Angstrom), which is longer than the end-to-end length of the protein obtained from NMR spectroscopy (43 Angstrom). Second, the measured contour length per amino acid from five different methods (4.0 +/- 0.2 Angstrom) is longer than the end-to-end length obtained from the crystal structure (3.6 Angstrom). Our measurement of the contour length takes into account all the internal degrees of freedom of the polypeptide chain, whereas crystallography measures the end-to-end length within the "frozen" protein structure. Furthermore, the control of contour length and therefore the number of amino acids unraveled before reaching the disulfide bond (n) facilitates the test of the chain length dependence on the folding time (tau(F)). We find that both a power law scaling tau(F) lambda n(lambda) with lambda = 4.4, and an exponential scaling with n(0.6) fit the data range, in support of different protein-folding scenarios.