Identification and characterization of multidomain monothiol glutaredoxin 3 from diploblastic Hydra.

Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.

Cleavage of gasdermin by apoptotic caspases triggers pyroptosis restricting bacterial colonization in Hydra.

Gasdermin (GSDM) proteins, as the direct executors of pyroptosis, are structurally and functionally conserved among vertebrates and play crucial roles in host defense against infection, inflammation, and cancer. However, the origin of functional GSDMs remains elusive in the animal kingdom. Here, we found that functional GSDME homologs first appeared in the cnidarian. Moreover, these animal GSDME homologs share evolutionarily conserved apoptotic caspase cleavage sites. Thus, we verified the functional conservation of apoptotic caspase-GSDME cascade in Hydra, a representative species of cnidarian. Unlike vertebrate GSDME homologs, HyGSDME could be cleaved by four Hydra caspase homologs with caspase-3 activity at two sites. Furthermore, in vivo activation of Hydra caspases resulted in HyGSDME cleavage to induce pyroptosis, exacerbating injury and restricting bacterial burden, which protects Hydra from pathogen invasion. In conclusion, these results suggest that GSDME-dependent pyroptosis may be an ancient and conserved host defense mechanism, which may contribute to better understanding on the origin and evolution of GSDMs.

Hydra gasdermin-gated pyroptosis signalling regulates tissue regeneration.

Pyroptosis, an inflammatory form of programmed cell death, is directly executed by gasdermin (GSDM) depending on its N-terminal pore-forming fragment-mediated membrane-disrupting, triggering intracellular contents release, which plays important roles in mammalian anti-infection and anti-tumor immune responses. However, whether pyroptosis engages in the regulation of tissue regeneration remains largely unknown. Here, utilizing Hydra vulgaris as the research model, we found that an HyCARD2-HyGSDME-mediated pyroptosis signalling is activated in both head and foot regenerated tips after amputation. Impeding pyroptosis by knocking down the expression of either HyGSDME or HyCARD2 significantly hampered both head and foot regeneration in Hydra. Mechanistically, the activation of HyCARD2-HyGSDME axis at wound sites is dependent of intracellular mitochondrial reactive oxygen species (mtROS), the removing of which hindered Hydra head regeneration. Moreover, the HyCARD2-HyGSDME axis-gated pyroptosis was found to enhance the initial secretion and upregulated expression of Wnt3. Collectively, these findings indicate that gasdermin-gated pyroptosis is critical for the evoking of Wnt signalling to facilitate Hydra tissue regeneration, which provides insights into functional diversification within the gasdermin family in the animal kingdom.

Glutaredoxin 1 from Evolutionary Ancient Hydra: Characteristics of the Enzyme and Its Possible Functions in Cell.

Glutaredoxin (Grx) is an antioxidant redox protein that uses glutathione (GSH) as an electron donor. Grx plays a crucial role in various cellular processes, such as antioxidant defense, control of cellular redox state, redox control of transcription, reversible S-glutathionylation of specific proteins, apoptosis, cell differentiation, etc. In the current study, we have isolated and characterized dithiol glutaredoxin from Hydra vulgaris Ind-Pune (HvGrx1). Sequence analysis showed that HvGrx1 belongs to the Grx family with the classical Grx motif (CPYC). Phylogenetic analysis and homology modeling revealed that HvGrx1 is closely related to Grx2 from zebrafish. HvGrx1 gene was cloned and expressed in Escherichia coli cells; the purified protein had a molecular weight of 11.82 kDa. HvGrx1 efficiently reduced ß-hydroxyethyl disulfide (HED) with the temperature optimum of 25°C and pH optimum 8.0. HvGrx1 was ubiquitously expressed in all body parts of Hydra. Expression of HvGrx1 mRNA and enzymatic activity of HvGrx1 were significantly upregulated post H2O2 treatment. When expressed in human cells, HvGrx1 protected the cells from oxidative stress and enhanced cell proliferation and migration. Although Hydra is a simple invertebrate, HvGrx1 is evolutionary closer to its homologs from higher vertebrates (similar to many other Hydra proteins).

A chromosome-scale epigenetic map of the Hydra genome reveals conserved regulators of cell state.

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best-characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra, including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris, an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large kilobase-scale chromatin interaction domains in the Hydra genome that contain transcriptionally coregulated genes. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types: isorhiza-type nematocytes and somatic gonad ectoderm. Finally, we identified novel candidate regulators of cell type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica more than 400 million years ago.

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra.

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Combining RNAi-Mediated ß-Catenin Inhibition and Reaggregation to Study Hydra Whole-Body Regeneration.

In addition to its ability to regenerate any amputated body part, the Hydra freshwater polyp shows the amazing ability to regenerate as a full polyp after a complete dissociation of its tissues. The developmental processes at work in reaggregates undergoing whole-body regeneration can be investigated at the molecular level by RNA interference (RNAi). Here we provide a protocol that combines ß-catenin RNAi with reaggregation. This protocol serves as a basis to generate "RNAi-reaggregates," followed by the extraction of high-quality RNA for the precise quantification of gene expression by real-time PCR. This protocol is efficient, providing both a molecular signature, with the significant downregulation of ß-catenin and Wnt3, as well as a robust phenotype, the lack of axis formation, which is observed in all reaggregates.

Cloning and characterization of Thioredoxin 1 from the Cnidarian Hydra.

Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site Cys-Gly-Pro-Cys and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH 7.0 and 25°C, respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in Escherichia coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.

Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra.

Here we discuss the developmental and homeostatic conditions necessary for Hydra regeneration. Hydra is characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. Intact Hydra polyps also exhibit a dynamic patterning along the oral-aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In aging Hydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

Ancestral role of TNF-R pathway in cell differentiation in the basal metazoan Hydra.

Tumour necrosis factor receptors (TNF-Rs) and their ligands, tumour necrosis factors, are highly conserved proteins described in all metazoan phyla. They function as inducers of extrinsic apoptotic signalling and facilitate inflammation, differentiation and cell survival. TNF-Rs use distinct adaptor molecules to activate signalling cascades. Fas-associated protein with death domain (FADD) family adaptors often mediate apoptosis, and TNF-R-associated factor (TRAF) family adaptors mediate cell differentiation and inflammation. Most of these pathway components are conserved in cnidarians, and, here, we investigated the Hydra TNF-R. We report that it is related to the ectodysplasin receptor, which is involved in epithelial cell differentiation in mammals. In Hydra, it is localised in epithelial cells with incorporated nematocytes in tentacles and body column, indicating a similar function. Further experiments suggest that it interacts with the Hydra homologue of a TRAF adaptor, but not with FADD proteins. Hydra FADD proteins colocalised with Hydra caspases in death effector filaments and recruited caspases, suggesting that they are part of an apoptotic signalling pathway. Regulating epithelial cell differentiation via TRAF adaptors therefore seems to be an ancient function of TNF-Rs, whereas FADD-caspase interactions may be part of a separate apoptotic pathway.

Differential expression of BMP inhibitors gremlin and noggin in Hydra suggests distinct roles during budding and patterning of tentacles.

BACKGROUND: Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. RESULTS: We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3ß inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. CONCLUSIONS: Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

Bacteria- and temperature-regulated peptides modulate ß-catenin signaling in Hydra.

Animal development has traditionally been viewed as an autonomous process directed by the host genome. But, in many animals, biotic and abiotic cues, like temperature and bacterial colonizers, provide signals for multiple developmental steps. Hydra offers unique features to encode these complex interactions of developmental processes with biotic and abiotic factors, and we used it here to investigate the impact of bacterial colonizers and temperature on the pattern formation process. In Hydra, formation of the head organizer involves the canonical Wnt pathway. Treatment with alsterpaullone (ALP) results in acquiring characteristics of the head organizer in the body column. Intriguingly, germfree Hydra polyps are significantly more sensitive to ALP compared to control polyps. In addition to microbes, ß-catenin-dependent pattern formation is also affected by temperature. Gene expression analyses led to the identification of two small secreted peptides, named Eco1 and Eco2, being up-regulated in the response to both Curvibacter sp., the main bacterial colonizer of Hydra, and low temperatures. Loss-of-function experiments revealed that Eco peptides are involved in the regulation of pattern formation and have an antagonistic function to Wnt signaling in Hydra.

Cell Sorting in Hydra vulgaris Arises from Differing Capacities for Epithelialization between Cell Types.

Hydra vulgaris exhibits a remarkable capacity to reassemble its body plan from a disordered aggregate of cells. Reassembly begins by sorting two epithelial cell types, endoderm and ectoderm, into inner and outer layers, respectively. The cellular features and behaviors that distinguish ectodermal and endodermal lineages to drive sorting have not been fully elucidated. To dissect this process, we use micromanipulation to position single cells of diverse lineages on the surface of defined multicellular aggregates and monitor sorting outcomes by live imaging. Although sorting has previously been attributed to intrinsic differences between the epithelial lineages, we find that single cells of all lineages sort to the interior of ectodermal aggregates, including single ectodermal cells. This reveals that cells of the same lineage can adopt opposing positions when sorting as individuals or a collective. Ectodermal cell collectives adopt their position at the aggregate exterior by rapidly reforming an epithelium that engulfs cells adhered to its surface through a collective spreading behavior. In contrast, aggregated endodermal cells persistently lose epithelial features. These non-epithelialized aggregates, like isolated cells of all lineages, are adherent passengers for engulfment by the ectodermal epithelium. We find that collective spreading of the ectoderm and persistent de-epithelialization in the endoderm also arise during local wounding in Hydra, suggesting that Hydra's wound-healing and self-organization capabilities may employ similar mechanisms. Together, our data suggest that differing propensities for epithelialization can sort cell types into distinct compartments to build and restore complex tissue architecture.

The structural basis of Bcl-2 mediated cell death regulation in hydra.

Apoptosis is regulated by evolutionarily conserved signaling pathways to remove damaged, diseased or unwanted cells. Proteins homologous to the B-cell lymphoma 2 (Bcl-2) family of proteins, the primary arbiters of mitochondrially mediated apoptosis, are encoded by the cnidarian Hydra vulgaris. We mapped interactions between pro-survival and pro-apoptotic Bcl-2 proteins of H. vulgaris by affinity measurements between Hy-Bcl-2-4, the sole confirmed pro-survival Bcl-2 protein, with BH3 motif peptides of two Bcl-2 proteins from hydra that displayed pro-apoptotic activity, Hy-Bak1 and Hy-BH3-only-2, and the BH3 motif peptide of the predicted pro-apoptotic protein Hy-Bax. In addition to peptides from hydra encoded pro-apoptotic proteins, Hy-Bcl-2-4 also engaged BH3 motif peptides from multiple human pro-apoptotic Bcl-2 proteins. Reciprocally, human pro-survival Bcl-2 proteins Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1/Bfl-1 bound to BH3 spanning peptides from hydra encoded pro-apoptotic Hy-Bak1, Hy-BH3-only and Hy-Bax. The molecular details of the interactions were determined from crystal structures of Hy-Bcl-2-4 complexes with BH3 motif peptides of Hy-Bak1 and Hy-Bax. Our findings suggest that the Bcl-2 family in hydra may function in a manner analogous to the Bcl-2 family in humans, and less like the worm Caenorhabditis elegans where evolutionary gene deletion has simplified the apoptotic program. Combined, our results demonstrate the powerful conservation of the interaction pattern between hydra and human Bcl-2 family members. Furthermore, our data reveal mechanistic differences in the mode of binding between hydra and sponges such as Geodia cydonium, with hydra encoded Bcl-2 resembling the more promiscuous pro-apoptotic Bcl-2 members found in mammals compared with its sponge counterpart.

Alternative pathways control actomyosin contractility in epitheliomuscle cells during morphogenesis and body contraction.

In adult Hydra, epitheliomuscle cells form the monolayered ecto- and endodermal epithelia. Their basal myonemes function as a longitudinal and circular muscle, respectively. Based on the observation that a Rho/Rock pathway, controlling the cell shape changes during detachment of Hydra buds, is not involved in body movement, at least two actomyosin compartments must exist in these cells: a basal one for body movement and a cortical one for cell shape changes. We therefore analyzed the regional and subcellular localization of the Ser19-phosphorylated myosin regulatory light chain (pMLC20). Along the body column, pMLC20 was detected strongly in the basal myonemes and weakly in the apical cell compartments of ectodermal epitheliomuscle cells. In cells of the bud base undergoing morphogenesis, pMLC20 was localized to intracellular stress fibers as well as to the apical and additionally to the lateral cortical compartment. Pharmacological inhibition revealed that pMLC20 is induced in these compartments by at least two independent pathways. In myonemes, MLC is phosphorylated mainly by myosin light chain kinase (MLCK). In contrast, the cortical apical and lateral MLC phosphorylation in constricting ectodermal cells of the bud base is stimulated via the Rho/ROCK pathway.

Dynamic interactions within the host-associated microbiota cause tumor formation in the basal metazoan Hydra.

The extent to which disturbances in the resident microbiota can compromise an animal's health is poorly understood. Hydra is one of the evolutionary oldest animals with naturally occurring tumors. Here, we found a causal relationship between an environmental spirochete (Turneriella spec.) and tumorigenesis in Hydra. Unexpectedly, virulence of this pathogen requires the presence of Pseudomonas spec., a member of Hydra´s beneficial microbiome indicating that dynamic interactions between a resident bacterium and a pathogen cause tumor formation. The observation points to the crucial role of commensal bacteria in maintaining tissue homeostasis and adds support to the view that microbial community interactions are essential for disease. These findings in an organism that shares deep evolutionary connections with all animals have implications for our understanding of cancer.

A novel bilateral grafting technique for studying patterning in Hydra.

Control of patterning and the specification of body axes are fundamental aspects of animal development involving complex interactions between chemical, physical, and genetic signals. The freshwater polyp Hydra has long been recognized as a useful model system to address these questions due to its simple anatomy, optical transparency, and strong regenerative abilities, which enabled clever grafting experiments to alter and probe patterning. Reliable methods exist for the transplantation of small tissue pieces into the body column or the combination of sections cut perpendicular to the body axis, which can be used to examine oral-aboral gradients and axis induction potential of tissue fragments. However, existing methods do not allow researchers to probe questions of axis alignment and lateral information exchange. We therefore developed a technique to produce chimeric animals split longitudinally along the body axis of the animal by anesthetizing the animals with the terpene linalool and threading the donor pieces onto pairs of fine glass needles. Our novel approach can be applied to study questions in Hydra research that have thus far been inaccessible, including patterning processes acting perpendicular to the oral-aboral axis and the extent of lateral cell migration.

Temperature and insulin signaling regulate body size in Hydra by the Wnt and TGF-beta pathways.

How multicellular organisms assess and control their size is a fundamental question in biology, yet the molecular and genetic mechanisms that control organ or organism size remain largely unsolved. The freshwater polyp Hydra demonstrates a high capacity to adapt its body size to different temperatures. Here we identify the molecular mechanisms controlling this phenotypic plasticity and show that temperature-induced cell number changes are controlled by Wnt- and TGF-ß signaling. Further we show that insulin-like peptide receptor (INSR) and forkhead box protein O (FoxO) are important genetic drivers of size determination controlling the same developmental regulators. Thus, environmental and genetic factors directly affect developmental mechanisms in which cell number is the strongest determinant of body size. These findings identify the basic mechanisms as to how size is regulated on an organismic level and how phenotypic plasticity is integrated into conserved developmental pathways in an evolutionary informative model organism.

Apoptosis in Hydra: function of HyBcl-2 like 4 and proteins of the transmembrane BAX inhibitor motif (TMBIM) containing family.

Mechanisms of programmed cell death differ between animals, plants and fungi. In animals, apoptotic cell death depends on caspases and Bcl-2 family proteins. These protein families are only found in multicellular animals, including cnidarians, insects and mammals. In contrast, members of the TMBIM-family of transmembrane proteins are conserved across all eukaryotes. Sequence comparisons of cell death related proteins between phyla indicate strong conservation of the genes involved. However, often it is not known whether this is paralleled by conservation of function. Here we present the first study to support an anti-apoptotic function of Bcl-2 like proteins in the cnidarian Hydra within a physiological context. We used transgenic Hydra expressing GFP-tagged HyBcl-2-like 4 protein in epithelial cells. The protein was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis induced by either the PI(3) kinase inhibitor wortmannin or by starvation. Moreover, we identified members of the TMBIM-family in Hydra including HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4. Expressing these TMBIM-family members in Hydra and human HEK cells, we found HyBax-inhibitor-1 protein localised to ER-membranes and HyLifeguard-family members localised to the plasma membrane and Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from camptothecin induced apoptosis. This work illustrates that the investigated Bcl-2- and TMBIM-family members represent evolutionarily conserved mitochondrial, ER, Golgi and plasma membrane proteins with anti-apoptotic functions. The participation of ER and Golgi proteins in the regulation of programmed cell death might be a very ancient feature.