Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

BackgroundAnimal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution.ResultsWe estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression.ConclusionsQuantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity

Regulatory motif discovery using a population clustering evolutionary algorithm

This paper describes a novel evolutionary algorithm for regulatory motif discovery in DNA promoter sequences. The algorithm uses data clustering to logically distribute the evolving population across the search space. Mating then takes place within local regions of the population, promoting overall solution diversity and encouraging discovery of multiple solutions. Experiments using synthetic data sets have demonstrated the algorithm's capacity to find position frequency matrix models of known regulatory motifs in relatively long promoter sequences. These experiments have also shown the algorithm's ability to maintain diversity during search and discover multiple motifs within a single population. The utility of the algorithm for discovering motifs in real biological data is demonstrated by its ability to find meaningful motifs within muscle-specific regulatory sequences

Protein palmitoylation plays an important role in Trichomonas vaginalis adherence

The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. As an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host and a better understanding of this process is a prerequisite for the development of therapies to combat infection. In this sense, recent work has shown S-acylation as a key modification that regulates pathogenesis in different protozoan parasites. However, there are no reports indicating whether this post-translational modification is a mechanism operating in T. vaginalis. In order to study the extent and function of S-acylation in T. vaginalis biology, we undertook a proteomic study to profile the full scope of S-acylated proteins in this parasite and reported the identification of 363 proteins involved in a variety of biological processes such as protein transport, pathogenesis related and signaling, among others. Importantly, treatment of parasites with the palmitoylation inhibitor 2-bromopalmitate causes a significant decrease in parasite: Parasite aggregation as well as adherence to host cells suggesting that palmitoylation could be modifying proteins that are key regulators of Trichomonas vaginalis pathogenesis.Fil: Nievas, Yésica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Vashisht, Ajay A.. University of California; Estados UnidosFil: Corvi, Maria Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Metz, Sebastián Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Johnson, Patricia J. University of California; Estados UnidosFil: Wohlschlegel, James A.. University of California; Estados UnidosFil: de Miguel, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Intrinsic and Extrinsic Connections of Tet3 Dioxygenase with CXXC Zinc Finger Modules.

Tet proteins are emerging as major epigenetic modulators of cell fate and plasticity. However, little is known about how Tet proteins are targeted to selected genomic loci in distinct biological contexts. Previously, a CXXC-type zinc finger domain in Tet1 was shown to bind CpG-rich DNA sequences. Interestingly, in human and mouse the Tet2 and Tet3 genes are adjacent to Cxxc4 and Cxxc10-1, respectively. The CXXC domains encoded by these loci, together with those in Tet1 and Cxxc5, identify a distinct homology group within the CXXC domain family. Here we provide evidence for alternative mouse Tet3 transcripts including the Cxxc10-1 sequence (Tet3(CXXC)) and for an interaction between Tet3 and Cxxc4. In vitro Cxxc4 and the isolated CXXC domains of Tet1 and Tet3(CXXC) bind DNA substrates with similar preference towards the modification state of cytosine at a single CpG site. In vivo Tet1 and Tet3 isoforms with and without CXXC domain hydroxylate genomic 5-methylcytosine with similar activity. Relative transcript levels suggest that distinct ratios of Tet3(CXXC) isoforms and Tet3-Cxxc4 complex may be present in adult tissues. Our data suggest that variable association with CXXC modules may contribute to context specific functions of Tet proteins

A survey of DNA motif finding algorithms

Background: Unraveling the mechanisms that regulate gene expression is a major challenge in biology. An important task in this challenge is to identify regulatory elements, especially the binding sites in deoxyribonucleic acid (DNA) for transcription factors. These binding sites are short DNA segments that are called motifs. Recent advances in genome sequence availability and in high-throughput gene expression analysis technologies have allowed for the development of computational methods for motif finding. As a result, a large number of motif finding algorithms have been implemented and applied to various motif models over the past decade. This survey reviews the latest developments in DNA motif finding algorithms.Results: Earlier algorithms use promoter sequences of coregulated genes from single genome and search for statistically overrepresented motifs. Recent algorithms are designed to use phylogenetic footprinting or orthologous sequences and also an integrated approach where promoter sequences of coregulated genes and phylogenetic footprinting are used. All the algorithms studied have been reported to correctly detect the motifs that have been previously detected by laboratory experimental approaches, and some algorithms were able to find novel motifs. However, most of these motif finding algorithms have been shown to work successfully in yeast and other lower organisms, but perform significantly worse in higher organisms.Conclusion: Despite considerable efforts to date, DNA motif finding remains a complex challenge for biologists and computer scientists. Researchers have taken many different approaches in developing motif discovery tools and the progress made in this area of research is very encouraging. Performance comparison of different motif finding tools and identification of the best tools have proven to be a difficult task because tools are designed based on algorithms and motif models that are diverse and complex and our incomplete understanding of the biology of regulatory mechanism does not always provide adequate evaluation of underlying algorithms over motif models.Peer reviewedComputer Scienc

DISCRETIZED GEOMETRIC APPROACHES TO THE ANALYSIS OF PROTEIN STRUCTURES

Proteins play crucial roles in a variety of biological processes. While we know that their amino acid sequence determines their structure, which in turn determines their function, we do not know why particular sequences fold into particular structures. My work focuses on discretized geometric descriptions of protein structure—conceptualizing native structure space as composed of mostly discrete, geometrically defined fragments—to better understand the patterns underlying why particular sequence elements correspond to particular structure elements. This discretized geometric approach is applied to multiple levels of protein structure, from conceptualizing contacts between residues as interactions between discrete structural elements to treating protein structures as an assembly of discrete fragments. My earlier work focused on better understanding inter-residue contacts and estimating their energies statistically. By scoring structures with energies derived from a stricter notion of contact, I show that native protein structures can be identified out of a set of decoy structures more often than when using energies derived from traditional definitions of contact and how this has implications for the evaluation of predictions that rely on structurally defined contacts for validation. Demonstrating how useful simple geometric descriptors of structure can be, I then show that these energies identify native structures on par with well-validated, detailed, atomistic energy functions. Moving to a higher level of structure, in my later work I demonstrate that discretized, geometrically defined structural fragments make good objects for the interactive assembly of protein backbones and present a software application which lets users do so. Finally, I use these fragments to generate structure-conditioned statistical energies, generalizing the classic idea of contact energies by incorporating specific structural context, enabling these energies to reflect the interaction geometries they come from. These structure-conditioned energies contain more information about native sequence preferences, correlate more highly with experimentally determined energies, and show that pairwise sequence preferences are tightly coupled to their structural context. Considered jointly, these projects highlight the degree to which protein structures and the interactions they comprise can be understood as geometric elements coming together in finely tuned ways

Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.

Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder

Xenopus: An ideal system for chemical genetics

Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches. genesis 50:207–218, 2012. © 2012 Wiley Periodicals, Inc

Transcriptional changes and regulation of voltage gated calcium channels across human stem cell differentiation

Human neural development comprises a phase during which a risk for psychiatric conditions such as schizophrenia is established. Robust evidence has linked voltage-gated calcium channels (VGCCs) to psychiatric illness. However, the biology of VGCCs in the context of human neural development, and particularly the prenatal stages, has not been sufficiently studied. This thesis examines the transcriptional regulation of VGCCs in a model system of neural development, the differentiation of human induced pluripotent stem cells (hiPSCs) into neurons. By applying bioinformatics tools to hiPSC and hiPSC-derived neuron data from dozens of donors, I characterized changes in the abundance and splicing of VGCCs across hiPSC differentiation into neurons. Additionally, motif-based bioinformatics tools were applied to identify candidate regulators of VGCC splicing and abundance. The analyses revealed that as hiPSCs differentiate into neurons, VGCC transcript levels robustly increase. The alternative splicing of CACNA1C, CACNA2D1, and CACNA2D2, including a cell state-specific switch in the utilization of Exons 21 and 22 in CACNA1C, were identified. Analyzing the regions of interest using two different bioinformatics tools identified a binding site for BRUNOL4 and BRUNOL5 upstream of CACNA1C Exon 21, which showed neither evolutionary conservation nor acceleration; and a binding site for HNRNPL near CACNA2D2 Exon 26 showing evolutionary acceleration in primates. Finally, transcription factor (TF) footprinting analyses of ATAC-seq data identified candidate TFs that might regulate multiple VGCC loci, several of which are expressed more highly in human fetal compared with postnatal brain development, including KLF7 and TCF4. Altogether, the results provide a foundation for future research investigating the functions of the identified RBPs and TFs in VGCC transcriptional regulation. The findings also indicate that hiPSCs possess the potential for providing insights into the changes that occur in VGCC transcription and putative mechanisms of VGCC regulation in human neural development and different cell states

Epigenetics and cell death: DNA hypermethylation in programmed retinal cell death.

BackgroundVertebrate genomes undergo epigenetic reprogramming during development and disease. Emerging evidence suggests that DNA methylation plays a key role in cell fate determination in the retina. Despite extensive studies of the programmed cell death that occurs during retinal development and degeneration, little is known about how DNA methylation might regulate neuronal cell death in the retina.MethodsThe developing chicken retina and the rd1 and rhodopsin-GFP mouse models of retinal degeneration were used to investigate programmed cell death during retinal development and degeneration. Changes in DNA methylation were determined by immunohistochemistry using antibodies against 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).ResultsPunctate patterns of hypermethylation paralleled patterns of caspase3-dependent apoptotic cell death previously reported to occur during development in the chicken retina. Degenerating rd1 mouse retinas, at time points corresponding to the peak of rod cell death, showed elevated signals for 5mC and 5hmC in photoreceptors throughout the retina, with the most intense staining observed in the peripheral retina. Hypermethylation of photoreceptors in rd1 mice was associated with TUNEL and PAR staining and appeared to be cCaspase3-independent. After peak rod degeneration, during the period of cone death, occasional hypermethylation was observed in the outer nuclear layer.ConclusionThe finding that cell-specific increases of 5mC and 5hmC immunostaining are associated with the death of retinal neurons during both development and degeneration suggests that changes in DNA methylation may play a role in modulating gene expression during the process of retinal degeneration. During retinal development, hypermethylation of retinal neurons associates with classical caspase-dependent apoptosis as well as caspase-3 independent cell death, while hypermethylation in the rd1 mouse photoreceptors is primarily associated with caspase-3 independent programmed cell death. These findings suggest a previously unrecognized role for epigenetic mechanisms in the onset and/or progression of programed cell death in the retina