Differential interactions between Beclin 1 and Bcl-2 family members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x(L), Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x(L) was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x(L). Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x(L). Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x(L) and Beclin 1.     

Extracellular N-domain alone can mediate specific heterophilic adhesion between members of the carcinoembryonic antigen family, CEACAM6 and CEACAM8

The domain(s) responsible for the specific heterophilic adhesion between two members of the carcinoembryonic antigen (CEA) family, CEACAM6 and CEACAM8, both of which with three extracellular domains, were investigated using Chinese hamster ovary (CHO) transfectants expressing chimeric antigens. Using a chimeric antigen in which the N-domain, a sole extracellular domain, of CEACAM3 was substituted with that of CEACAM6, it was shown that the N-domain of CEACAM6 alone was able to mediate specific adhesion to CEACAM8. Furthermore, the chimeric antigen was shown to bind significantly to chimeric CEA whose N-domain was substituted with that of CEACAM8, but not to unsubstituted CEA. These results demonstrate that the N-domain alone is sufficient and other domains of CEACAM6 or CEACAM8 are not required for this specific binding. We therefore propose a model of heterophilic interaction between the N-domains, which is distinct from that of CEA-CEA homophilic binding.     

Complementary expression and neurite outgrowth activity of netrin-G subfamily members.

Classical members of the UNC6/netrin family are secreted proteins which play a role as long-range cues for directing growth cones. We here identified in mice a novel member netrin-G2 which constitute a subfamily with netrin-G1 among the UNC6/netrin family. Both of these netrin-Gs are characterized by glycosyl phosphatidyl-inositol linkage onto cells, molecular variants presumably generated by alternative splicing and lack of any appreciable affinity to receptors for classical netrins. These genes are preferentially expressed in the central nervous system with complementary distribution in most brain areas, that is netrin-G1 in the dorsal thalamus, olfactory bulb and inferior colliculus, and netrin-G2 in the cerebral cortex, habenular nucleus and superior colliculus. Consistently, immunohistochemical analysis revealed that netrin-G1 molecules are present on thalamocortical but not corticothalamic axons. Thalamic and neocortical neurons extended long neurites on immobilized recombinant netrin-G1 or netrin-G2 in vitro. Immobilized anti-netrin-G1 antibodies altered shapes of cultured thalamic neurons. We propose that netrin-Gs provide short-range cues for axonal and/or dendritic behavior through bi-directional signaling.     

Two members of the IgLON family are expressed in a restricted region of the developing chick brain and neural crest

The precise expression patterns of two IgLON genes, CEPU-1 and limbic system-associated membrane protein (LAMP), were studied during early embryogenesis. It was found that expression of both was localized to restricted regions of the brain and neural crest. In the developing neural tube, CEPU-1 was expressed in the isthmus and a restricted region of the hindbrain, whereas LAMP was expressed in the anterior midbrain. Most neural crest cells expressed LAMP, whereas CEPU-1 expression was limited to crest cells derived from the hindbrain. These results suggest that members of the IgLON family have important roles during embryogenesis, particularly in brain formation and differentiation.     

Developmental expression and biochemical characterization of Emu family members

Kidney development has often served as a model for epithelial-mesenchymal cell interaction where the branching epithelium of the ureteric bud induces the metanephrogenic mesenchyme to form epithelial nephrons. In a screen for genes differentially expressed during kidney development, we have identified a novel gene that is dynamically expressed in the branching ureter and the developing nephrons. It was designated Emu1 since it shares an N-terminal cysteine-rich domain with Emilin1/2 and Multimerin. This highly conserved EMI domain is also found in another novel protein (Emu2) of similar protein structure: an N-terminal signal peptide followed by the EMI domain, an interrupted collagen stretch, and a conserved C-terminal domain of unknown function. We identified two further secreted EMI domain proteins, prompting us to compare their gene and protein structures, the EMI domain phylogeny, as well as the embryonic expression pattern of known (Emilin1/2, Multimerin) and novel (Emu1/2, Emilin3, Multimerin2) Emu gene family members. Emu1 and Emu2 not only show a similar structural organization, but furthermore a striking complementary expression in organs developing through epithelial-mesenchymal interactions. In these tissues, Emu1 is restricted to epithelial and Emu2 to mesenchymal cells. Preliminary biochemical analysis of Emu1/2 confirmed that they are secreted glycoproteins which are attached to the extracellular matrix and capable of forming homo- and heteromers via disulfide bonding. The widespread, but individually distinct expression patterns of all Emu gene family members suggest multiple functions during mouse embryogenesis. Their multidomain protein structure may indicate that Emu proteins interact with several different extracellular matrix components and serve to connect and integrate the function of multiple partner molecules.     

Transgenic analysis of the Smad family of TGF-beta signal transducers in Drosophila melanogaster suggests new roles and new interactions between family members

Smad signal transducers are required for transforming growth factor-beta-mediated developmental events in many organisms including humans. However, the roles of individual human Smad genes (hSmads) in development are largely unknown. Our hypothesis is that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad). We expressed six hSmad and four dSmad transgenes in Drosophila melanogaster using the Gal4/UAS system and compared their phenotypes. Phylogenetically related human and Drosophila Smads induced similar phenotypes supporting the hypothesis. In contrast, two nearly identical hSmads generated distinct phenotypes. When expressed in wing imaginal disks, hSmad2 induced oversize wings while hSmad3 induced cell death. This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles. Our observations also suggest new roles for the dSmads, Med and Dad, in dActivin signaling and potential interactions between these family members. Overall, the study demonstrates that transgenic methods in Drosophila can provide new information about non-Drosophila members of developmentally important multigene families.     

Detecting and characterizing gene conversions between multigene family members

We used a variety of methods to detect known gene conversions in the actin gene families of five angiosperm species, the beta-globin gene families of two primate species, and the Zfx/Zfy gene families of seven mammalian species. Our goal was to devise a working strategy which would allow the analysis of the members of a multigene family in order to determine whether there had been gene conversions between its members, identify the genes involved in the gene conversions, establish the lengths of the converted regions, and determine the polarities of the gene conversions. We show that three phylogenetic methods and the homoplasy test of Maynard Smith and Smith perform relatively poorly on our data sets because the sequences we analyzed had large levels of multiple substitutions. The method of Sawyer, the compatibility method of Jakobsen and Easteal, the partition matrix method of Jakobsen, Wilson, and Easteal, and the co-double method of Balding, Nichols, and Hunt can be used to identify the genes which have been involved in gene conversions. The co-double method is more powerful than other methods but requires orthologous sequences from related species. Compatibility, phylogenetic, and nucleotide substitution distribution statistics methods can be used to identify the location of the converted region(s). Site-by-site compatibility analyses can also be used to identify the direction of the conversion event(s). Combinations of these methods can therefore be used to establish the presence, locations, and polarities of gene conversions between multigene family members.     

Difficult relationships--interactions between family members and staff in long-term care

Staff of long-term care facilities and family members have a common responsibility to ensure the best course of treatment and everyday care for residents who often cannot speak for themselves. Understanding the difference between instrumental and preservative care, and who the proper agent is to provide care in each category will not only improve staff/family interactions, but residential care in general. The Resident Enrichment and Activity Program improves the family/staff relationship obliquely by involving family in social activities; the Family Involvement in Care program, and the Patterns in Caregiving program directly target the relationship and involve the facility's administration to effect policy change.     

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth

The IgLONs are a family of glycosyl phosphatidyl inositol-linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell-cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell-cell adhesion and recognition.     

Distinct developmental expression of two elongase family members in zebrafish

Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.     

RTP family members induce functional expression of mammalian odorant receptors

Transport of G protein-coupled receptors (GPCRs) to the cell surface membrane is critical in order for the receptors to recognize their ligands. However, mammalian GPCR odorant receptors (ORs), when heterologously expressed in cells, are poorly expressed on the cell surface. Here we show that the transmembrane proteins RTP1 and RTP2 promote functional cell surface expression of ORs expressed in HEK293T cells. Genes encoding these proteins are expressed specifically in olfactory neurons. These proteins are associated with OR proteins and enhance the OR responses to odorants. Similar although weaker effects were seen with a third protein, REEP1. These findings suggest that RTP1 and RTP2 in particular play significant roles in the translocation of ORs to the plasma membrane as well as in the functioning of ORs. We have used this approach to identify active odorant ligands for ORs, providing a platform for screening the chemical selectivity of the large OR family.     

Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-beta family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-alpha/beta (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.     

Gene and cluster-specific expression of the Iroquois family members during mouse development

Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.     

Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins

MADS domain (for M CM1, A G, D EFA and S RF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled-coil structure suggests a role in establishing a protein—protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C-terminal region of AG in a yeast two-hybrid selection, 156 clones that encode potential AG-interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.