The regulation of programmed and pathological cell death in C. elegans

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2007.Includes bibliographical references.Programmed cell death, or apoptosis, is important in the development and homeostasis of metazoans. In the nematode C. elegans, four genes, egl-1, ced-9, ced-4, and ced-3, constitute the core pathway acting in all somatic programmed cell deaths. This pathway is evolutionarily conserved in humans. The BH3-only protein EGL-1 is transcriptionally upregulated in cells fated to undergo programmed cell death, and EGL-1 blocks cell-death inhibition by the cell-death regulator CED-9, a Bcl-2 family member. The binding of EGL- 1 to CED-9 releases the Apaf- 1-like adaptor protein CED-4 from CED-9, so that CED-4 can activate the caspase CED-3, a protease that is the effector of programmed cell death. In this thesis, I describe three projects, each of which examines one aspect of C. elegans cell death. From. screens for mutations that increase cell death in a sensitized genetic background, I identified a gene that protects cells from programmed cell death.(cont.) This gene, spk-1, encodes a homolog of SR protein kinases, which regulate alternative splicing. Previous work has shown that ced-4 pre-mRNA is alternatively spliced to generate two transcripts that function oppositely in cell death. I found that spk-1 regulates ced-4 transcript splicing, thereby influencing the amount of programmed cell death that occurs. From a screen for genes that promote programmed cell death, I isolated a mutation in a conserved non-coding element in the transcriptionally regulated cell-death activator gene egl-1. This element regulates the deaths of specific cells in the C. elegans ventral nervous system. I found a novel C. elegans transcription factor, Y38C9A. 1, that binds this element and might function to regulate egl-1 transcription and programmed cell death in the ventral nervous system. In addition to the programmed cell deaths that occur in C. elegans, pathological death of specific cells can be caused by mutations in some genes. I characterized two genes, lin-24 and lin-33, that can mutate to cause the inappropriate death of specific hypodermal blast cells. One of these genes, lin-24, contains a domain similar to that found in some bacterial toxins.(cont.) By morphological and genetic criteria, I show that the lin-24- and lin-33-mediated deaths are unlike previously characterized necrotic and apoptotic cell deaths in C. elegans. These deaths require some of the genes responsible for engulfing the corpses generated by programmed cell death, even though the deaths do not require the core genes of the genetic pathway of programmed cell death.by Brendan D. Galvin.Ph.D

A target repurposing approach identifies N-myristoyltransferase as a new candidate drug target in filarial nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC50 values of 2.5-10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis

A High-Quality De novo Genome Assembly from a Single Mosquito Using PacBio Sequencing.

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life

SPK-1, an SR protein kinase, inhibits programmed cell death in Caenorhabditis elegans

To identify genes involved in protecting cells from programmed cell death in Caenorhabditis elegans, we performed a genetic screen to isolate mutations that cause an increase in the number of programmed cell deaths. We screened for suppressors of the cell-death defect caused by a partial loss-of-function mutation in ced-4, which encodes an Apaf-1 homolog that promotes programmed cell death by activating the caspase CED-3. We identified one extragenic ced-4 suppressor, which has a mutation in the gene spk-1. The spk-1 gene encodes a protein homologous to serine-arginine-rich (SR) protein kinases, which are thought to regulate splicing. Previous work suggests that ced-4 can be alternatively spliced and that the splice variants function oppositely, with the longer transcript (ced-4L) inhibiting programmed cell death. spk-1 might promote cell survival by increasing the amount of the protective ced-4L splice variant. We conclude that programmed cell death in C. elegans is regulated by an alternative splicing event controlled by the SR protein kinase SPK-1.Howard Hughes Medical Institute (Predoctoral Fellowship)Damon Runyon Cancer Research Foundation (Postdoctoral Fellowship)Charles A. King Trust Postdoctoral Fellowship Progra

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor

Structures of the NMT inhibitors, DDD85646 and DDD100870.

<p>Structures of the NMT inhibitors, DDD85646 and DDD100870.</p