The SH3 Domain of UNC-89 (obscurin) Interacts with Paramyosin, a Coiled-coil Protein, in Caenorhabditis Elegans Muscle

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website

Novel Cell Penetrating Peptide-adaptors Effect Intracellular Delivery and Endosomal Escape of Protein Cargos

The use of cell penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high affinity noncovalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression

A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations.

Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this 'endosomal escape problem.' Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells

Kinetic and affinity constants for CPP-adaptor binding to CBS-myoglobin (or TIP-myoglobin).

<p>Kinetic and affinity constants for CPP-adaptor binding to CBS-myoglobin (or TIP-myoglobin).</p

Biolayer interferometry analysis of CPP-adaptor-cargo binding.

<p>Association (0-300s) and dissociation (300-600s) phases are shown for A, TAT-CaM; B, TAT-CALL3; C, SAP-CaM; D, SAP(E)-CaM and E, TAT-Tropo binding to CBS-myoglobin (A-D) or TIP-myoglogbin. Analyte concentrations were 1000 nM (green), 500 nM (magenta), 250 nM (orange), 125 nM (red), and 63 nM (blue). Fits are shown to a single-state global model for which the constants are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178648#pone.0178648.t001" target="_blank">Table 1</a>. F, EDTA dissociation phases for 1000 nM analyte samples moved into EDTA after dissociation phase. Binding normalized to % specific binding to eliminate differences in amplitude, allowing direct comparisons.</p

Cell penetration assay for a variety of CPPs and adaptor proteins for delivery of myoglobin to BHK cells.

<p>Each panel are images from experiments with different CPP-adaptors: A, TAT-CaM 2.0; B, CALML3; C, SAP-CaM; D, SAP(E)-CaM. BHK cells were treated for 1 h with DyLight 550 fluorescently labeled CBS-myo (rendered as white in left panels, red in center and right panels), in either the absence or presence of CPP-adaptor, washed and imaged live. Center images are optical sections set at a similar depth of the nucleus (NucBlue staining, white, center and right panels), as determined by position within the Z-stack. Orthogonal projections are shown at the right (boxed in red) and top (boxed in green) sides of each panel. Cytoplasmic compartments in live cells were visualized using CellTracker Green CMFDA dye (green in right panels). Comparison of CPP-adaptor-treated versus untreated cells indicates that in all cases, myoglobin was delivered and localized primarily to the cytoplasm. Scale bars in all panels, 20 μm.</p