Identification of a novel gene family involved in osmotic stress response in Caenorhabditis elegans

Organisms exposed to the damaging effects of high osmolarity accumulate solutes to increase cytoplasmic osmolarity. Yeast accumulates glycerol in response to osmotic stress, activated primarily by MAP kinase Hog1 signaling. A pathway regulated by protein kinase C (PKC1) also responds to changes in osmolarity and cell wall integrity. C. elegans accumulates glycerol when exposed to high osmolarity, but the molecular pathways responsible for this are not well understood. We report the identification of two genes, osm-7 and osm-11, which are related members of a novel gene family. Mutations in either gene lead to high internal levels of glycerol and cause an osmotic resistance phenotype (Osr). These mutants also have an altered defecation rhythm (Dec). Mutations in cuticle collagen genes dpy-2, dpy-7, and dpy-10 cause a similar Osr Dec phenotype. osm-7 is expressed in the hypodermis and may be secreted. We hypothesize that osm-7 and osm-11 interact with the cuticle, and disruption of the cuticle causes activation of signaling pathways that increase glycerol production. The phenotypes of osm-7 are not suppressed by mutations in MAP kinase or PKC pathways, suggesting that C. elegans uses signaling pathways different from yeast to mount a response to osmotic stress.     

MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans

The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydrolysis of acyl-CoA lipid esters. The mechanisms by which these lipid esters are directed to the appropriate membranes in vivo, and their precise roles in vesicle biogenesis, are not yet understood. Here, we present the first report on membrane associated ACBP domain-containing protein-1 (MAA-1), a novel membrane-associated member of the acyl-CoA-binding protein family. We show that in Caenorhabditis elegans, MAA-1 localizes to intracellular membrane organelles in the secretory and endocytic pathway and that mutations in maa-1 reduce the rate of endosomal recycling. A lack of maa-1 activity causes a change in endosomal morphology. Although in wild type, many endosomal organelles have long tubular protrusions, loss of MAA-1 activity results in loss of the tubular domains, suggesting the maa-1 is required for the generation or maintenance of these domains. Furthermore, we demonstrate that MAA-1 binds fatty acyl-CoA in vitro and that this ligand-binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA-dependent process during vesicle formation.     

A Caenorhabditis elegans MAP kinase kinase, MEK-1, is involved in stress responses

The c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, was shown to be involved in the response to various stresses in cultured cells. However, there is little in vivo evidence indicating a role for a JNK pathway in the stress response of an organism. We identified the Caenorhabditis elegans mek-1 gene, which encodes a 347 amino acid protein highly homologous to mammalian MKK7, an activator of JNK. Mek-1 reporter fusion proteins are expressed in pharyngeal muscle, uterus, a portion of intestine, and neurons. A mek-1 deletion mutant is hypersensitive to copper and cadmium ions and to starvation. A wild-type mek-1 transgene rescued the hypersensitivity to the metal ions. Double mutants of mek-1 with an eat-5, eat-11 or eat-18 mutation, which are characterized by a limited feeding defect, showed distinct growth defects under normal conditions. Expression of an activated form of MEK-1 in the whole animal or specifically in the pharynx inhibited pharyngeal pumping. These results suggest a role for mek-1 in stress responses, with a focus in the pharynx and/or intestine.     

A survival pathway for Caenorhabditis elegans with a blocked unfolded protein response

The unfolded protein response (UPR) counteracts stress caused by unprocessed ER client proteins. A genome-wide survey showed impaired induction of many UPR target genes in xbp-1 mutant Caenorhabditis elegans that are unable to signal in the highly conserved IRE1-dependent UPR pathway. However a family of genes, abu (activated in blocked UPR), was induced to higher levels in ER-stressed xbp-1 mutant animals than in ER-stressed wild-type animals. RNA-mediated interference (RNAi) inactivation of a representative abu family member, abu-1 (AC3.3), activated the ER stress marker hsp-4::gfp in otherwise normal animals and killed 50% of ER-stressed ire-1 and xbp-1 mutant animals. Abu-1(RNAi) also enhanced the effect of inactivation of sel-1, an ER-associated protein degradation gene. The nine abu genes encode highly related type I transmembrane proteins whose lumenal domains have sequence similarity to a mammalian cell surface scavenger receptor of endothelial cells that binds chemically modified extracellular proteins and directs their lysosomal degradation. Our findings that ABU-1 is an intracellular protein located within the endomembrane system that is induced by ER stress in xbp-1 mutant animals suggest that ABU proteins may interact with abnormal ER client proteins and this function may be particularly important in animals with an impaired UPR.     

CUP-1 is a novel protein involved in dietary cholesterol uptake in Caenorhabditis elegans

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane "cholesterol recognition/interaction amino acid consensus" (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.     

Myotactin, a novel hypodermal protein involved in muscle-cell adhesion in Caenorhabditis elegans.

In C. elegans, assembly of hypodermal hemidesmosome-like structures called fibrous organelles is temporally and spatially coordinated with the assembly of the muscle contractile apparatus, suggesting that signals are exchanged between these cell types to position fibrous organelles correctly. Myotactin, a protein recognized by monoclonal antibody MH46, is a candidate for such a signaling molecule. The antigen, although expressed by hypodermis, first reflects the pattern of muscle elements and only later reflects the pattern of fibrous organelles. Confocal microscopy shows that in adult worms myotactin and fibrous organelles show coincident localization. Further, cell ablation studies show the bodywall muscle cells are necessary for normal myotactin distribution. To investigate myotactin's role in muscle-hypodermal signaling, we characterized the myotactin locus molecularly and genetically. Myotactin is a novel transmembrane protein of approximately 500 kd. The extracellular domain contains at least 32 fibronectin type III repeats and the cytoplasmic domain contains unique sequence. In mutants lacking myotactin, muscle cells detach when embryonic muscle contraction begins. Later in development, fibrous organelles become delocalized and are not restricted to regions of the hypodermis previously contacted by muscle. These results suggest myotactin helps maintain the association between the muscle contractile apparatus and hypodermal fibrous organelles.     

Differential requirement of unfolded protein response pathway for calreticulin expression in Caenorhabditis elegans

Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR) pathway, which increases the expression of chaperones to maintain the homeostasis. Calreticulin is a calcium-binding chaperone located in the lumen of endoplasmic reticulum (ER). Here we show that in response to a UPR inducing reagent, tunicamycin, the expression of calreticulin (crt-1) is specifically up-regulated in Caenorhabditis elegans. Tunicamycin (TM) induced expression of the crt-1 requires IRE-1 and XBP-1 but is ATF-6 and PEK-1 independent. Analysis of the crt-1 promoter reveals a putative XBP-1 binding site at the -284 to -278 bp region, which was shown to be necessary for TM-mediated induction. Genetic analysis of crt-1 mutants and mutants of UPR pathway genes show various degrees of developmental arrest upon TM treatment. Our results suggest that the TM-induced UPR pathway culminates in the up-regulation of crt-1, which protects the worm from deleterious accumulation of unfolded proteins in the ER. Knockdown of the crt-1, pdi-2, or pdi-3 increased the crt-1 expression, whereas knockdown of the hsp-3 or hsp-4 did not have any effect on crt-1 expression, indicating the existence of complex compensatory networks to cope up with ER stress.     

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPR^mt) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPR^mt, suggesting that the UPR^mt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPR^mt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPR^mt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPR^mt and longevity.     

SKN-1/Nrf,stress responses,and aging in Caenorhabditis elegans

The mammalian Nrf/CNC proteins (Nrf1, Nrf2, Nrf3, p45 NF-E2) perform a wide range of cellular protective and maintenance functions. The most thoroughly described of these proteins, Nrf2, is best known as a regulator of antioxidant and xenobiotic defense, but more recently has been implicated in additional functions that include proteostasis and metabolic regulation. In the nematode Caenorhabditis elegans, which offers many advantages for genetic analyses, the Nrf/CNC proteins are represented by their ortholog SKN-1. Although SKN-1 has diverged in aspects of how it binds DNA, it exhibits remarkable functional conservation with Nrf/CNC proteins in other species and regulates many of the same target gene families. C. elegans may therefore have considerable predictive value as a discovery model for understanding how mammalian Nrf/CNC proteins function and are regulated in vivo. Work in C. elegans indicates that SKN-1 regulation is surprisingly complex and is influenced by numerous growth, nutrient, and metabolic signals. SKN-1 is also involved in a wide range of homeostatic functions that extend well beyond the canonical Nrf2 function in responses to acute stress. Importantly, SKN-1 plays a central role in diverse genetic and pharmacologic interventions that promote C. elegans longevity, suggesting that mechanisms regulated by SKN-1 may be of conserved importance in aging. These C. elegans studies predict that mammalian Nrf/CNC protein functions and regulation may be similarly complex and that the proteins and processes that they regulate are likely to have a major influence on mammalian life- and healthspan.     

The Caenorhabditis elegans MAPK phosphatase VHP-1 mediates a novel JNK-like signaling pathway in stress response

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP-regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp-1 gene, encoding an MKP that acts preferentially on the c-Jun N-terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp-1 are arrested during larval development. This vhp-1 defect is suppressed by loss-of-function mutations in the kgb-1, mek-1, and mlk-1 genes encoding a JNK-like MAPK, an MKK7-type MAPKK, and an MLK-type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP-1 in the KGB-1 pathway. Loss-of-function mutations in each component in the KGB-1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP-1 plays a pivotal role in the integration and fine-tuning of the stress response regulated by the KGB-1 MAPK pathway.     

RMD-1, a novel microtubule-associated protein, functions in chromosome segregation in Caenorhabditis elegans

For proper chromosome segregation, the sister kinetochores must attach to microtubules extending from the opposite spindle poles. Any errors in microtubule attachment can induce aneuploidy. In this study, we identify a novel conserved Caenorhabditis elegans microtubule-associated protein, regulator of microtubule dynamics 1 (RMD-1), that localizes to spindle microtubules and spindle poles. Depletion of RMD-1 induces severe defects in chromosome segregation, probably through merotelic attachments between microtubules and chromosomes. Although rmd-1 embryos also have a mild defect in microtubule growth, we find that mutants of the microtubule growth regulator XMAP215/ZYG-9 show much weaker segregation defects. This suggests that the microtubule growth defect in rmd-1 embryos does not cause abnormal chromosome segregation. We also see that RMD-1 interacts with aurora B in vitro. Our results suggest that RMD-1 functions in chromosome segregation in C. elegans embryos, possibly through the aurora B–mediated pathway. Human homologues of RMD-1 could also bind microtubules, which would suggest a function for these proteins in chromosome segregation during mitosis in other organisms as well.     

Zinc homeostasis is involved in unfolded protein response under salt stress

Accumulation of unfolded protein or misfolded protein causes endoplasmic reticulum (ER) stress. Increased salt concentration activates a stress response pathway in the ER in Arabidopsis thaliana to induce the expression of several salt stress response genes, leading to a more optimal protein folding environment in the ER. In addition, some salt stress-regulated proteins require zinc for their activity, including some zinc-dependent DNA binding proteins and zinc-finger proteins. In a recent study, we reported that ZTP29, a putative zinc transporter at the ER membrane, is involved in the response to salt stress through regulation of zinc level in the ER to induce the UPR pathway. In this addendum, we propose a testable hypothesis for the role of ZTP29 in the response to salt stress via the regulation of zinc levels in the ER.Key words: zinc, ER stress, unfolded protein response, salt stress, arabidopsisHigh salinity is a common abiotic stress that adversely affects plant growth and crop production.¹ Plants must sense the stress and transduce stress signals to activate response pathways leading to adaptation to, or tolerance of, the abiotic stress in salt environment.² Salt stress activates a stress response pathway in the endoplasmic reticulum (ER) in Arabidopsis thaliana, indicating that the adaptation of plants to salt stress involves ER stress signal regulation.^3,4 There is limited understanding of molecular mechanisms on ER stress in plants, as compared to yeast and mammalian cells. bZIP60, bZIP28, bZIP17 are three membrane-associated basic domain/leucine zipper (bZIP) factors, which have been reported as candidates for ER-folding proteins in plants.^5–7 BiP acts as a general chaperone in the ER lumen, due to its ability to discriminate between properly folded and unfolded protein structures.⁸ Unfolded or misfolded proteins are retained in the ER and form stable complexes with BiP and other ER resident chaperones.⁹ Zinc deficiency induces unfolded protein response (UPR) in most eukaryotes.¹⁰ Zinc is an important trace element, which participates in physiological and biochemical process in vivo. The requirement of zinc for proper ER function is evolutionarily conserved.     

Conserved domains subserve novel mechanisms and functions in DKF-1, a Caenorhabditis elegans protein kinase D

Protein kinase D (PKD) isoforms are effectors in signaling pathways controlled by diacylglycerol. PKDs contain conserved diacylglycerol binding (C1a, C1b), pleckstrin homology (PH), and Ser/Thr kinase domains. However, the properties of conserved domains may vary within the context of distinct PKD polypeptides. Such functional/structural malleability (plasticity) was explored by studying Caenorhabditis elegans D kinase family-1 (DKF-1), a PKD that governs locomotion in vivo. Phorbol ester binding with C1b alone activates classical PKDs by relieving C1-mediated inhibition. In contrast, C1a avidly ligated phorbol 12-myristate 13-acetate (PMA) and anchored DKF-1 at the plasma membrane. C1b bound PMA (moderate affinity) and cooperated with C1a in targeting DKF-1 to membranes. Mutations at a "Pro(11)" position in C1 domains were inactivating; kinase activity was minimal at PMA concentrations that stimulated wild type DKF-1 approximately 10-fold. DKF-1 mutants exhibited unchanged, maximum kinase activity after cells were incubated with high PMA concentrations. Titration in situ revealed that translocation and activation of wild type and mutant DKF-1 were tightly and quantitatively linked at all PMA concentrations. Thus, C1 domains positively regulated phosphotransferase activity by docking DKF-1 with pools of activating lipid. A PH domain inhibits kinase activity in classical PKDs. The DKF-1 PH module neither inhibited catalytic activity nor bound phosphoinositides. Consequently, the PH module is an obligatory, positive regulator of DKF-1 activity that is compromised by mutation of Lys(298) or Trp(396). Phosphorylation of Thr(588) switched on DKF-1 kinase activity. Persistent phosphorylation of Thr(588) (activation loop) promoted ubiquitinylation and proteasome-mediated degradation of DKF-1. Each DKF-1 domain displayed novel properties indicative of functional malleability (plasticity).     

Caenorhabditis elegans gelsolin-like protein 1 is a novel actin filament-severing protein with four gelsolin-like repeats

The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins.     

ER stress and the unfolded protein response

Conformational diseases are caused by mutations altering the folding pathway or final conformation of a protein. Many conformational diseases are caused by mutations in secretory proteins and reach from metabolic diseases, e.g. diabetes, to developmental and neurological diseases, e.g. Alzheimer's disease. Expression of mutant proteins disrupts protein folding in the endoplasmic reticulum (ER), causes ER stress, and activates a signaling network called the unfolded protein response (UPR). The UPR increases the biosynthetic capacity of the secretory pathway through upregulation of ER chaperone and foldase expression. In addition, the UPR decreases the biosynthetic burden of the secretory pathway by downregulating expression of genes encoding secreted proteins. Here we review our current understanding of how an unfolded protein signal is generated, sensed, transmitted across the ER membrane, and how downstream events in this stress response are regulated. We propose a model in which the activity of UPR signaling pathways reflects the biosynthetic activity of the ER. We summarize data that shows that this information is integrated into control of cellular events, which were previously not considered to be under control of ER signaling pathways, e.g. execution of differentiation and starvation programs.     

Direct observation of stress response in Caenorhabditis elegans using a reporter transgene

Transgenic Caenorhabditis elegans expressing jellyfish Green Fluorescent Protein under the control of the promoter for the inducible small heat shock protein gene hsp-16-2 have been constructed. Transgene expression parallels that of the endogenous hsp-16 gene, and, therefore, allows direct visualization, localization, and quantitation of hsp-16 expression in living animals. In addition to the expected upregulation by heat shock, we show that a variety of stresses, including exposure to superoxide-generating redox-cycling quinones and the expression of the human beta amyloid peptide, specifically induce the reporter transgene. The quinone induction is suppressed by coincubation with L-ascorbate. The ability to directly observe the stress response in living animals significantly simplifies the identification of both exogenous treatments and genetic alterations that modulate stress response, and possibly life span, in C. elegans.