Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein.

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice.

BACKGROUND The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4+CD25+ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. METHODS/FINDINGS Key parameters for infection outcome in E. multilocularis-infected fgl2-/- (AE-fgl2-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4+CD25+ Treg suspensions were incubated with CD4+ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. CONCLUSIONS FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.

Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa)

Scope: Today, about 2–8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Methods and results: Sera from 42 Spanish lettuce-allergic patients were obtained from pa-tients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Conclusion: Two new major lettuce allergens—a thaumatin-like protein and an aspartyl protease—have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

SIRE-1, a copia/Ty1-like retroelement from soybean, encodes a retroviral envelope-like protein

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large, copia/Ty1-like retroelement designated SIRE-1. A copy of this element has been recovered from a Glycine max genomic library. DNA sequence analysis of two SIRE-1 subclones revealed that SIRE-1 contains a long, uninterrupted, ORF between the 3′ end of the pol ORF and the 3′ long terminal repeat (LTR), a region that harbors the env gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, α-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of SIRE-1 to the copia/Ty1 family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to copia/Ty1 retrotransposons.

Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1

Posttranslational modifications such as ubiquitination and phosphorylation play an important role in the regulation of cellular protein function. Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the recently identified family of nuclear protein kinases that act as corepressors for homeodomain transcription factors. Here, we show that HIPK2 is regulated by a ubiquitin-like protein, SUMO-1. We demonstrate that HIPK2 localizes to nuclear speckles (dots) by means of a speckle-retention signal. This speckle-retention signal contains a domain that interacts with a mouse ubiquitin-like protein conjugating (E2) enzyme, mUBC9. In cultured cells, HIPK2 is covalently modified by SUMO-1, and the SUMO-1 modification of HIPK2 correlates with its localization to nuclear speckles (dots). Thus, our results provide firm evidence that the nuclear protein kinase HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles (dots).

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression

Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. In this study, we report the discovery of a new B. subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids. Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh. Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate. We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage. In a B. subtilis ptsH1 mutant strain, synthesis of β-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR. Additional disruption of the crh gene caused almost complete relief from CCR. In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of β-xylosidase was also completely relieved from glucose repression. These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46.

Peropsin, a novel visual pigment-like protein located in the apical microvilli of the retinal pigment epithelium

A visual pigment-like protein, referred to as peropsin, has been identified by large-scale sequencing of cDNAs derived from human ocular tissues. The corresponding mRNA was found only in the eye, where it is localized to the retinal pigment epithelium (RPE). Peropsin immunoreactivity, visualized by light and electron microscopy, localizes the protein to the apical face of the RPE, and most prominently to the microvilli that surround the photoreceptor outer segments. These observations suggest that peropsin may play a role in RPE physiology either by detecting light directly or by monitoring the concentration of retinoids or other photoreceptor-derived compounds.

An HP1-like protein is missing from transcriptionally silent micronuclei of Tetrahymena

We report the identification and cloning of a 28-kDa polypeptide (p28) in Tetrahymena macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from Drosophila. Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix–turn–helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

Rump white inversion in the mouse disrupts dipeptidyl aminopeptidase-like protein 6 and causes dysregulation of Kit expression

The mouse rump white (Rw) mutation causes a pigmentation defect in heterozygotes and embryonic lethality in homozygotes. At embryonic day (E) 7.5, Rw/Rw embryos are retarded in growth, fail to complete neurulation and die around E 9.5. The Rw mutation is associated with a chromosomal inversion spanning 30 cM of the proximal portion of mouse chromosome 5. The Rw embryonic lethality is complemented by the W19H deletion, which spans the distal boundary of the Rw inversion, suggesting that the Rw lethality is not caused by the disruption of a gene at the distal end of the inversion. Here, we report the molecular characterization of sequences disrupted by both inversion breakpoints. These studies indicate that the distal breakpoint of the inversion is associated with ectopic Kit expression and therefore may be responsible for the dominant pigmentation defect in Rw/+ mice; whereas the recessive lethality of Rw is probably due to the disruption of the gene encoding dipeptidyl aminopeptidase-like protein 6, Dpp6 [Wada, K., Yokotani, N., Hunter, C., Doi, K., Wenthold, R. J. & Shimasaki, S. (1992) Proc. Natl. Acad. Sci. USA 89, 197–201] located at the proximal inversion breakpoint.

The amphiphysin-like protein 1 (ALP1) interacts functionally with the cABL tyrosine kinase and may play a role in cytoskeletal regulation

cABL is a protooncogene, activated in a subset of human leukemias, whose protein product is a nonreceptor tyrosine kinase of unknown function. cABL has a complex structure that includes several domains and motifs found in proteins implicated in signal transduction pathways. An approach to elucidate cABL function is to identify proteins that interact directly with cABL and that may serve as regulators or effectors of its activity. To this end, a protein-interaction screen of a phage expression library was undertaken to identify proteins that interact with specific domains of cABL. An SH3-domain-containing protein has been identified that interacts with sequences in the cABL carboxyl terminus. The cDNA encoding ALP1 (amphiphysin-like protein 1) was isolated from a 16-day mouse embryo. ALP1 has high homology to BIN1, a recently cloned myc-interacting protein, and also shows significant homology to amphiphysin, a neuronal protein cloned from human and chicken. The amino terminus has homology to two yeast proteins, Rvs167 and Rvs161, which are involved in cell entry into stationary phase and cytoskeletal organization. ALP1 binds cABL in vitro and in vivo. Expression of ALP1 results in morphological transformation of NIH 3T3 fibroblasts in a cABL-dependent manner. The properties of ALP1 suggest that it may be involved in possible cytoskeletal functions of the cABL kinase. Additionally, these results provide further evidence for the importance of the cABL carboxyl terminus and its binding proteins in the regulation of cABL function.

A PII-like protein in Arabidopsis: Putative role in nitrogen sensing

PII is a protein allosteric effector in Escherichia coli and other bacteria that indirectly regulates glutamine synthetase at the transcriptional and post-translational levels in response to nitrogen availability. Data supporting the notion that plants have a nitrogen regulatory system(s) includes previous studies showing that the levels of mRNA for plant nitrogen assimilatory genes such as glutamine synthetase (GLN) and asparagine synthetase (ASN) are modulated by carbon and organic nitrogen metabolites. Here, we have characterized a PII homolog (GLB1) in two higher plants, Arabidopsis thaliana and Ricinus communis (Castor bean). Each plant PII-like protein has high overall identity to E. coli PII (50%). Western blot analyses reveal that the plant PII-like protein is a nuclear-encoded chloroplast protein. The PII-like protein of plants appears to be regulated at the transcriptional level in that levels of GLB1 mRNA are affected by light and metabolites. To initiate studies of the in vivo function of the Arabidopsis PII-like protein, we have constructed transgenic lines in which PII expression is uncoupled from its native regulation. Analyses of these transgenic plants support the notion that the plant PII-like protein may serve as part of a complex signal transduction network involved in perceiving the status of carbon and organic nitrogen. Thus, the PII protein found in archaea, bacteria, and now in higher eukaryotes (plants) is one of the most widespread regulatory proteins known, providing evidence for an ancestral metabolic regulatory mechanism that may have existed before the divergence of these three domains of life.

Glucose-induced Autophagy of Peroxisomes in Pichia pastoris Requires a Unique E1-like Protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Δ had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(ΔATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25–30 kDa protein. This conjugate was not observed in cells expressing Gsa7(ΔATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.