Silva N et al. (2016) J Cell Biol "UNC-84: "LINC-ing" chromosome movement and double strand break repair."

Silva N, Jantsch V

[J Cell Biol, 2016]

Assaults to our DNA take place at a high frequency and are incompatible with life. In this issue, Lawrence et al. (2016. J. Cell Biol https://doi.org/10.1083/jcb.201604112) demonstrate that a novel complex links the nucleus with cytoplasmic microtubules for the promotion of DNA repair by homologous recombination.

Silva, N. et al. (2015) International Worm Meeting "Restriction of Topoisomerase II levels by Aminopeptidase P prevents genome instability."

Isaac, E.R., Martinez-Perez, E., Boulton, S.J., Barroso, C., Brooks, D., Matsuzaki, K., Silva, N.

[International Worm Meeting, 2015]

Topoisomerase II (Top2) is an essential enzyme that binds DNA to produce a transient double strand break (DSB) that allows the release of topological stress (supercoiling) and catenations that arise mostly during DNA mitotic replication. Since Top2 binds covalently to DNA, the amount of DNA-bound Top2 must be tightly controlled as these complexes impede the progression of DNA polymerases, causing replication fork collapse, unscheduled DSB formation and genome instability. In fact, Top2 poisons that prevent Top2 removal from DNA, such as etoposide, are key components of anticancer drugs. However, the mechanisms that regulate the amount of DNA-bound Top2 are not understood. We show that in C. elegans and human cells lacking aminopeptidase P (APP-1/ (XPNPEP1 in humans), which removes the N-terminal amino acid of peptides carrying a proline in position 2, display an increase in cellular levels of Top2, demonstrating that APP-1 is a key regulator of Top2 levels. Importantly, this overall increase in Top2 levels causes accumulation of Top2 on DNA in humans and worms, suggesting that Top2 activity is modulated through the control of its cellular levels. app-1 mutant worms display reduced fertility, genomic instability, and a dramatic accumulation of RAD-51 recombination intermediates in their germlines. RAD-51 intermediates persist even in the absence of SPO-11, the topoisomerase-like enzyme that induces DSBs during meiosis to promote inter-homologue crossover events, demonstrating that APP-1 prevents the formation of unscheduled DSBs in the germline. Knockdown of XPNPEP1 in human cells also causes formation of unscheduled DSBs. Analysis of transgenic strains demonstrates that the enzymatic activity of APP-1 is required for genome stability and to prevent TOP-2 accumulation, while analysis of DNA repair in app-1 mutants reveals the presence of replication-related damage, suggesting that excess TOP-2 impairs the progression of DNA polymerases. In fact, app-1 mutants are hypersensitive to etoposide, but resistant to top-2 knockdown by RNAi, confirming that they accumulate functional TOP-2. Thus, APP-1 is a conserved regulator of Top2 levels and increased amounts of Top2 cause genome instability. These findings could have therapeutic implications, since increasing the cellular levels of Top2 should sensitize cells to Top2 poisons commonly used to treat a variety of human cancers.

Silva N et al. (2022) PLoS Genet "Proline-specific aminopeptidase P prevents replication-associated genome instability."

Barroso C, Matsuzaki K, Martinez-Perez E, Brooks DR, Isaac RE, Roman-Trufero M, Silva N, Craig H, Boulton SJ, Castellano-Pozo M

[PLoS Genet, 2022]

Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1's role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.

Silva N et al. (2013) Cell Death Differ "Pro-crossover factors regulate damage-dependent apoptosis in the Caenorhabditis elegans germ line."

Santonicola P, Adamo A, La Volpe A, Silva N, Martinez-Perez E

[Cell Death Differ, 2013]

During meiosis, DNA double-strand breaks (DSBs) are physiologically induced to start the recombination process and promote the formation of interhomologue crossovers (COs), which are required to ensure faithful chromosome segregation into the gametes. The timely repair of DSBs is an essential part of the meiotic programme, as accumulation of unprocessed DSBs during the pachytene stage of meiotic prophase triggers a DNA damage checkpoint response that induces apoptosis of damaged cells. We show that CO-promoting factors MSH-4, MSH-5, and ZHP-3, but not COSA-1, are required for the apoptotic response of the meiotic DNA damage checkpoint. Lack of MSH-4 or MSH-5 suppresses the apoptotic response observed in some DNA repair-defective mutants such as fcd-2 and brc-1 (orthologues of FANCD2 and BRCA1), irrespectively of the amount of DSBs present in pachytene nuclei. Although ionizing radiation fails to induce apoptosis in msh-4/5-mutant backgrounds, it induces transcriptional activation of the apoptosis-activator egl-1, which is controlled by the Caenorhabditis elegans p53 orthologue CEP-1. This finding suggests that MSH-4/5 involvement in the apoptotic response occurs downstream or independently of damage sensing and checkpoint activation. This study establishes a role for pro-CO factors MSH-4/5 and ZHP-3 in the execution of apoptosis at late meiotic prophase following the accumulation of exogenous or endogenous DNA damage.

Silva N et al. (2014) Dev Cell "The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression."

Tognetti S, Snijders AP, Encheva V, Ferrandiz N, Lightfoot J, Martinez-Perez E, Hanni S, Faull P, Furger A, Silva N, Speck C, Telecan O, Barroso C

[Dev Cell, 2014]

Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology, and poorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly. Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homolog pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology-independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of Mad2 in the spindle assembly checkpoint.

Vader G et al. (2014) Dev Cell "HORMA domains at the heart of meiotic chromosome dynamics."

Musacchio A, Vader G

[Dev Cell, 2014]

HORMA domain proteins are required for the careful orchestration of chromosomal organization during meiosis. Kim et al. (2014) and Silva et al. (2014) now provide structural and functional insights into the roles of C. elegans HORMA proteins, revealing parallels to the function of the HORMA protein MAD2 in mitotic checkpoint signaling.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Ge YL et al. (2002) Anticancer Res "Effects of adenoviral gene transfer of C. elegans n-3 fatty acid desaturase on the lipid profile and growth of human breast cancer cells."

Kang ZB, Chen Z, Leaf A, Kang JX, Cluette-Brown J, Laposata M, Ge YL

[Anticancer Res, 2002]

Background: Current evidence from both experimental and human studies indicates that omega-6 polyunsaturated fatty acids (n-6 PUFAs) promote breast tumor development, whereas long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) exert suppressive effects. The ratio of n-6 to n-3 fatty acids appears to be an important factor in controlling tumor development. Human cells usually have a very high n-6/n-3 fatty acid ratio because they cannot convert n-6 PUFAs to n-3 PUFAs due to lack of an n-3 desaturase found in C. elegans. Materials and Methods: Adenoviral strategies were used to introduce the C. elegans fat-1 gene encoding an n-3 fatty acid desaturase into human breast cancer cells followed by examination of the n-6/n-3 fatty acid ratio and growth of the cells. Results: Infection of MCF-7 cells with an adenovirus carrying the fat-1 gene resulted in a high expression of the n-3 fatty acid desaturase. Lipid analysis indicated a remarkable increase in the levels of n-3 PUFAs accompanied with a large decrease in the contents of n-6 PUFAs, leading to a change of the n-6/n-3 ratio from 12.0 to 0.8. Accordingly, production of the eicosanoids derived from n-6 PUFA was reduced significantly in cells expressing the fat-1 gene. Importantly, the gene transfer induced mass cell death and inhibited cell proliferation. Conclusion: The gene transfer of the n-3 fatty acid desaturase, as a novel approach, can effectively modify the n-6/n-3 fatty acid ratio of human tumor cells and provide an anticancer effect, without the need of exogenous n-3 PUFA supplementation. These data also increase the understanding of the effects of n-3 fatty acids and the n-6/n-3 ratio on cancer prevention and treatment.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.

Kang ZB et al. (2001) Proc Natl Acad Sci U S A "Adenoviral gene transfer of Caenorhabditis elegans n--3 fatty acid desaturase optimizes fatty acid composition in mammalian cells."

Leaf A, Laposata M, Kang ZB, Kang JX, Chen Z, Ge Y, Cluette-Brown J

[Proc Natl Acad Sci U S A, 2001]

Omega-3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n-3 PUFAs is insufficient in most Western people, whereas the level of n-6 PUFAs is relatively too high, with an n-6/n-3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n-3 PUFAs in mammals relies on chronic dietary intake of fat rich in n-3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n-3 PUFAs or to convert n-6 to n-3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n - 3 PUFA contents and dramatically balance the ratio of n-6/n-3 PUFAs, Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n-6 PUFAs to the corresponding n-3 PUFAs, and changed the n-6/n-3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n-6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.