Ashkavand Z et al. (2019) Aging Cell "Corrupted ER-mitochondrial calcium homeostasis promotes the collapse of proteostasis."

Sarasija S, Ryan KC, Laboy JT, Norman KR, Ashkavand Z

[Aging Cell, 2019]

Aging and age-related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C.elegans presenilin cause elevated ER to mitochondria calciumsignaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C.elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.

Ketel, CS et al. (2005) Mol Cell Biol "Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes."

Vargas, ML, Andersen, EF, Strome, S, Ketel, CS, Suh, J, Simon, JA

[Mol Cell Biol, 2005]

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is > 1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

Wan G et al. (2021) EMBO J "ZSP-1 is a Z granule surface protein required for Z granule fluidity and germline immortality in Caenorhabditis elegans."

Pagano D, Dodson AE, Bajaj L, Kennedy S, Fei Y, Wan G, Fields B

[EMBO J, 2021]

Germ granules are biomolecular condensates that form in germ cells of all/most animals, where they regulate mRNA expression to promote germ cell function and totipotency. In the adult Caenorhabditis elegans germ cell, these granules are composed of at least four distinct sub-compartments, one of which is the Z granule. To better understand the role of the Z granule in germ cell biology, we conducted a genetic screen for genes specifically required for Z granule assembly or morphology. Here, we show that zsp-1, which encodes a low-complexity/polyampholyte-domain protein, is required for Z granule homeostasis. ZSP-1 localizes to the outer surface of Z granules. In the absence of ZSP-1, Z granules swell to an abnormal size, fail to segregate with germline blastomeres during development, and lose their liquid-like character. Finally, ZSP-1 promotes piRNA- and siRNA-directed gene regulation and germline immortality. Our data suggest that Z granules coordinate small RNA-based gene regulation to promote germ cell function and that ZSP-1 helps/is need to maintain Z granule morphology and liquidity.

Krishna P et al. (1988) J Biol Chem "Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA."

Krishna P, Kennedy BP, van de Sande JH, McGhee JD

[J Biol Chem, 1988]

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.

Gorgon S et al. (2024) Cancer Res Commun "A drug discovery pipeline for MAPK/ERK pathway inhibitors in C. elegans."

Gorgon S, Hemmingsson O, Billing O, Eriksson AU

[Cancer Res Commun, 2024]

Oncogenic signaling through the MAPK/ERK pathway drives tumor progression in many cancers. While targeted MAPK/ERK pathway inhibitors improve survival in selected patients, most tumors are resistant. New drugs could be identified in small animal models that, unlike in vitro models, can address oral uptake, compound bioavailability and toxicity. This requires pharmacological conformity between human and model MAPK/ERK pathways, and available phenotypic assays. Here, we test if the conserved MAPK/ERK pathway in C. elegans could serve as a model for pharmacological inhibition and we develop in vivo pipelines for high throughput compound screens. Using fluorescence-based image analysis of vulva development as a readout for MAPK/ERK activity, we obtained excellent assay Z-scores for the MEK inhibitors trametinib (Z = 0.95), mirdametinib (Z = 0.93), and AZD8330 (Z = 0.87), as well as the ERK inhibitor temuterkib (Z = 0.86). The throughput was 800 wells per hour, with an average seed density of 25.5 animals per well. Readouts included drug efficacy, toxicity, and pathway specificity, which was tested against pathway activating upstream (lin-15)- and downstream (lin-1) mutants. To validate the model in a high throughput setting, we screened a blinded library of 433 anti-cancer compounds and identified four MEK inhibitors among seven positive hits. Our results highlight a high degree of pharmacological conformity between C. elegans and human MAPK/ERK pathways and the presented high-throughput pipeline may discover and characterize novel inhibitors in vivo.

Henderson JR et al. (2003) Cell Motil Cytoskeleton "ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes."

Pomies P, Beckerle MC, Auffray C, Henderson JR

[Cell Motil Cytoskeleton, 2003]

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.

Ryan KC et al. (2024) Aging Dis "C. elegans Presenilin Mediates Inter-Organelle Contacts and Communication that Is Required for Lysosome Activity."

Barroso M, Ryan KC, Norman KR, Ashkavand Z, Wang L, Laboy JT

[Aging Dis, 2024]

Compromised lysosome function is implicated in the pathology of many neurodegenerative diseases, including Alzheimer's disease (AD). Familial Alzheimer's disease (fAD) is caused primarily by mutations in the presenilin encoding genes, but the underlying mechanism remains obscure. Loss of the conserved C. elegans presenilin orthologue SEL-12 results in increased mitochondrial calcium, which promotes neurodegeneration. Here, we find that sel-12 mutant lysosomes, independent of SEL-12 proteolytic activity, are significantly enlarged and more alkaline due to increased ER-to-mitochondrial calcium signaling and concomitant mitochondrial oxidative stress. These defects and their dependence on mitochondrial calcium are recapitulated in human fAD fibroblasts, demonstrating a conserved role for mitochondrial calcium in presenilin-mediated lysosome dysfunction. sel-12 mutants also have increased contact surface area between the ER, mitochondria, and lysosomes, suggesting sel-12 has an additional role in modulating organelle contact and communication. Overall, we demonstrate that SEL-12 maintains lysosome acidity and lysosome health by controlling ER-to-mitochondrial calcium signaling.

Farkas Z et al. (2019) FASEB J "The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin."

Budai Z, Mehta A, Herit F, Shakir MA, Petric M, Zhou Z, Rajnavolgyi E, Takacs-Vellai K, Boissan M, Sandor S, Orban TI, Farkas Z, Kovacs T, Szondy Z, Jung SY, Niedergang F, Liu X, Qin J, Bajtay Z

[FASEB J, 2019]

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (<i>Caenorhabditis elegans</i>/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavolgyi, E., Szondy, Z., Budai, Z., Orban, T. I., Sandor, S., Mehta, A., Bajtay, Z., Kovacs, T., Jung, S. Y., Shakir, M. A., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takacs-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

Mi-Mi L et al. (2012) J Cell Biol "Z-line formins promote contractile lattice growth and maintenance in striated muscles of C. elegans."

Mi-Mi L, Kemphues K, Pruyne D, Votra S, Bretscher A

[J Cell Biol, 2012]

Muscle contraction depends on interactions between actin and myosin filaments organized into sarcomeres, but the mechanism by which actin filaments incorporate into sarcomeres remains unclear. We have found that, during larval development in Caenorhabditis elegans, two members of the actin-assembling formin family, CYK-1 and FHOD-1, are present in striated body wall muscles near or on sarcomere Z lines, where barbed ends of actin filaments are anchored. Depletion of either formin during this period stunted growth of the striated contractile lattice, whereas their simultaneous reduction profoundly diminished lattice size and number of striations per muscle cell. CYK-1 persisted at Z lines in adulthood, and its near complete depletion from adults triggered phenotypes ranging from partial loss of Z line-associated filamentous actin to collapse of the contractile lattice. These results are, to our knowledge, the first genetic evidence implicating sarcomere-associated formins in the in vivo organization of the muscle cytoskeleton.

Ryan KC et al. (2021) Aging Cell "Increased mitochondrial calcium uptake and concomitant mitochondrial activity by presenilin loss promotes mTORC1 signaling to drive neurodegeneration."

Laboy JT, Ryan KC, Norman KR, Sarasija S, Ashkavand Z, Samarakoon R

[Aging Cell, 2021]

Metabolic dysfunction and protein aggregation are common characteristics that occur in age-related neurodegenerative disease. However, the mechanisms underlying these abnormalities remain poorly understood. We have found that mutations in the gene encoding presenilin in Caenorhabditis elegans, sel-12, results in elevated mitochondrial activity that drives oxidative stress and neuronal dysfunction. Mutations in the human presenilin genes are the primary cause of familial Alzheimer's disease. Here, we demonstrate that loss of SEL-12/presenilin results in the hyperactivation of the mTORC1 pathway. This hyperactivation is caused by elevated mitochondrial calcium influx and, likely, the associated increase in mitochondrial activity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL-12 function. Consistent with high mTORC1 activity, we find that SEL-12loss reduces autophagosome formation, and this reduction is prevented by limiting mitochondrial calcium uptake. Moreover, the improvements of proteostasis and neuronal defects in sel-12mutants due to mTORC1 inhibition require the induction of autophagy. These results indicate that mTORC1hyperactivation exacerbates the defects in proteostasis and neuronal function in sel-12mutants and demonstrate a critical role of presenilin in promoting neuronal health.