ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues.Peer reviewe

Discrete Kinetic Models from Funneled Energy Landscape Simulations

A general method for facilitating the interpretation of computer simulations of protein folding with minimally frustrated energy landscapes is detailed and applied to a designed ankyrin repeat protein (4ANK). In the method, groups of residues are assigned to foldons and these foldons are used to map the conformational space of the protein onto a set of discrete macrobasins. The free energies of the individual macrobasins are then calculated, informing practical kinetic analysis. Two simple assumptions about the universality of the rate for downhill transitions between macrobasins and the natural local connectivity between macrobasins lead to a scheme for predicting overall folding and unfolding rates, generating chevron plots under varying thermodynamic conditions, and inferring dominant kinetic folding pathways. To illustrate the approach, free energies of macrobasins were calculated from biased simulations of a non-additive structure-based model using two structurally motivated foldon definitions at the full and half ankyrin repeat resolutions. The calculated chevrons have features consistent with those measured in stopped flow chemical denaturation experiments. The dominant inferred folding pathway has an “inside-out”, nucleation-propagation like character

TbUNC119 and Its Binding Protein Complex Are Essential for Propagation, Motility, and Morphogenesis of Trypanosoma brucei Procyclic Form Cells

Flagellum-mediated motility of Trypanosoma brucei is considered to be essential for the parasite to complete stage development in the tsetse fly vector, while the mechanism by which flagellum-mediated motility is controlled are not fully understood. We thus compared T. brucei whole gene products (amino acid sequence) with Caenorhabditis elegans UNC (uncoordinated) proteins, in order to find uncharacterized motility-related T. brucei genes. Through in silico analysis, we found 88 gene products which were highly similar to C. elegans UNC proteins and categorized them as TbCEUN (T. brucei gene products which have high similarity to C. elegans UNC proteins). Approximately two thirds of the 88 TbCEUN gene products were kinesin-related molecules. A gene product highly similar to C. elegans UNC119 protein was designated as TbUNC119. RNAi-mediated depletion of TbUNC119 showed no apparent phenotype. However, knock-down analysis of both TbUNC119 and its binding protein (TbUNC119BP) which was found by yeast two-hybrid analysis showed characteristic phenotypes, including reduced motility, morphological change (extended cell shape), and cellular apoptosis. Based on the observed phenotypes, possible function of the TbUNC119 and TbUNC119BP is discussed

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies

The highly attenuated oncolytic recombinant vaccinia virus GLV-1h68: comparative genomic features and the contribution of F14.5L inactivation

As a new anticancer treatment option, vaccinia virus (VACV) has shown remarkable antitumor activities (oncolysis) in preclinical studies, but potential infection of other organs remains a safety concern. We present here genome comparisons between the de novo sequence of GLV-1h68, a recombinant VACV, and other VACVs. The identified differences in open reading frames (ORFs) include genes encoding host-range selection, virulence and immune modulation proteins, e.g., ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. Phylogenetic analyses indicate that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L-null and revertant viruses. GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism

Structure-Function Relationship of Cytoplasmic and Nuclear IκB Proteins: An In Silico Analysis

Cytoplasmic IκB proteins are primary regulators that interact with NF-κB subunits in the cytoplasm of unstimulated cells. Upon stimulation, these IκB proteins are rapidly degraded, thus allowing NF-κB to translocate into the nucleus and activate the transcription of genes encoding various immune mediators. Subsequent to translocation, nuclear IκB proteins play an important role in the regulation of NF-κB transcriptional activity by acting either as activators or inhibitors. To date, molecular basis for the binding of IκBα, IκBβ and IκBζ along with their partners is known; however, the activation and inhibition mechanism of the remaining IκB (IκBNS, IκBε and Bcl-3) proteins remains elusive. Moreover, even though IκB proteins are structurally similar, it is difficult to determine the exact specificities of IκB proteins towards their respective binding partners. The three-dimensional structures of IκBNS, IκBζ and IκBε were modeled. Subsequently, we used an explicit solvent method to perform detailed molecular dynamic simulations of these proteins along with their known crystal structures (IκBα, IκBβ and Bcl-3) in order to investigate the flexibility of the ankyrin repeat domains (ARDs). Furthermore, the refined models of IκBNS, IκBε and Bcl-3 were used for multiple protein-protein docking studies for the identification of IκBNS-p50/p50, IκBε-p50/p65 and Bcl-3-p50/p50 complexes in order to study the structural basis of their activation and inhibition. The docking experiments revealed that IκBε masked the nuclear localization signal (NLS) of the p50/p65 subunits, thereby preventing its translocation into the nucleus. For the Bcl-3- and IκBNS-p50/p50 complexes, the results show that Bcl-3 mediated transcription through its transactivation domain (TAD) while IκBNS inhibited transcription due to its lack of a TAD, which is consistent with biochemical studies. Additionally, the numbers of identified flexible residues were equal in number among all IκB proteins, although they were not conserved. This could be the primary reason for their binding partner specificities

The LRRK2 signalling system

The LRRK2 gene is a major contributor to genetic risk for Parkinson's disease and understanding the biology of the leucine-rich repeat kinase 2 (LRRK2, the protein product of this gene) is an important goal in Parkinson's research. LRRK2 is a multi-domain, multi-activity enzyme and has been implicated in a wide range of signalling events within the cell. Because of the complexities of the signal transduction pathways in which LRRK2 is involved, it has been challenging to generate a clear idea as to how mutations and disease associated variants in this gene are altered in disease. Understanding the events in which LRRK2 is involved at a systems level is therefore critical to fully understand the biology and pathobiology of this protein and is the subject of this review

Identification of a New Rhoptry Neck Complex RON9/RON10 in the Apicomplexa Parasite Toxoplasma gondii

Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes