C-terminal Domain Of Unknown Function Research Articles

Truncation of a novel C-terminal domain of a β-glucanase improves its thermal stability and specific activity.

Enzymes that degrade β-glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo-β-1,3(4)-glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum, was cloned and expressed in Pichia pastoris. Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C-terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR-mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A-ΔC, exhibited significantly enhanced thermal stability at 50°C, with D-values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A-ΔC demonstrated a 20%-25% increase in halotolerance at 1.25 and 1.5M NaCl, respectively, compared to the full-length enzymes. Notably, specific activity against cereal β-glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress-tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo-β-1,3(4)-glucanase. Additionally, it sheds light on a group of endo-β-1,3(4)-glucanases from Antarctic rock-inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function.

MUT-7 exoribonuclease activity and localization are mediated by an ancient domain.

The MUT-7 family of 3'-5' exoribonucleases is evolutionarily conserved across the animal kingdom and plays essential roles in small RNA production in the germline. Most MUT-7 homologues carry a C-terminal domain of unknown function named MUT7-C appended to the exoribonuclease domain. Our analysis shows that the MUT7-C is evolutionary ancient, as a minimal version of the domain exists as an individual protein in prokaryotes. In animals, MUT7-C has acquired an insertion that diverged during evolution, expanding its functions. Caenorhabditis elegans MUT-7 contains a specific insertion within MUT7-C, which allows binding to MUT-8 and, consequently, MUT-7 recruitment to germ granules. In addition, in C. elegans and human MUT-7, the MUT7-C domain contributes to RNA binding and is thereby crucial for ribonuclease activity. This RNA-binding function most likely represents the ancestral function of the MUT7-C domain. Overall, this study sheds light on MUT7-C and assigns two functions to this previously uncharacterized domain.

Insights into peculiar fungal LPMO family members holding a short C-terminal sequence reminiscent of phosphate binding motifs

Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature. White-rot Polyporales, which are major fungal wood decayers, may possess up to 60 LPMO-encoding genes belonging to the auxiliary activities family 9 (AA9). Yet, the functional relevance of such multiplicity remains to be uncovered. Previous comparative transcriptomic studies of six Polyporales fungi grown on cellulosic substrates had shown the overexpression of numerous AA9-encoding genes, including some holding a C-terminal domain of unknown function (“X282”). Here, after carrying out structural predictions and phylogenetic analyses, we selected and characterized six AA9-X282s with different C-term modularities and atypical features hitherto unreported. Unexpectedly, after screening a large array of conditions, these AA9-X282s showed only weak binding properties to cellulose, and low to no cellulolytic oxidative activity. Strikingly, proteomic analysis revealed the presence of multiple phosphorylated residues at the surface of these AA9-X282s, including a conserved residue next to the copper site. Further analyses focusing on a 9 residues glycine-rich C-term extension suggested that it could hold phosphate-binding properties. Our results question the involvement of these AA9 proteins in the degradation of plant cell wall and open new avenues as to the divergence of function of some AA9 members.

Arsenite Methyltransferase Diversity and Optimization of Methylation Efficiency.

Arsenic is methylated by arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferases (ArsMs). ArsM crystal structures show three domains (an N-terminal SAM binding domain (A domain), a central arsenic binding domain (B domain), and a C-terminal domain of unknown function (C domain)). In this study, we performed a comparative analysis of ArsMs and found a broad diversity in structural domains. The differences in the ArsM structure enable ArsMs to have a range of methylation efficiencies and substrate selectivities. Many small ArsMs with 240-300 amino acid residues have only A and B domains, represented by RpArsM from Rhodopseudomonas palustris. These small ArsMs have higher methylation activity than larger ArsMs with 320-400 residues such as Chlamydomonas reinhardtii CrArsM, which has A, B, and C domains. To examine the role of the C domain, the last 102 residues in CrArsM were deleted. This CrArsM truncation exhibited higher As(III) methylation activity than the wild-type enzyme, suggesting that the C-terminal domain has a role in modulating the rate of catalysis. In addition, the relationship of arsenite efflux systems and methylation was examined. Lower rates of efflux led to higher rates of methylation. Thus, the rate of methylation can be modulated in multiple ways.

Enhancing extracellular production of recombinant proteins in Escherichia coli by co-expressing with a haloarchaeal protein containing a putative LolA-like domain.

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by β-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli. A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli. KEY POINTS: • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.

A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm.

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

The C-Terminal Domain of the Sudan Ebolavirus L Protein Is Essential for RNA Binding and Methylation.

The large (L) protein of Ebola virus is a key protein for virus replication. Its N-terminal region harbors the RNA-dependent RNA polymerase activity, and its C terminus contains a cap assembling line composed of a capping domain and a methyltransferase domain (MTase) followed by a C-terminal domain (CTD) of unknown function. The L protein MTase catalyzes methylation at the 2'-O and N-7 positions of the cap structures. In addition, the MTase of Ebola virus can induce cap-independent internal adenosine 2'-O-methylation. In this work, we investigated the CTD role in the regulation of the cap-dependent and cap-independent MTase activities of the L protein. We found that the CTD, which is enriched in basic amino acids, plays a key role in RNA binding and in turn regulates the different MTase activities. We demonstrated that the mutation of CTD residues modulates specifically the different MTase activities. Altogether, our results highlight the pivotal role of the L protein CTD in the control of viral RNA methylation, which is critical for Ebola virus replication and escape from the innate response in infected cells.IMPORTANCE Ebola virus infects human and nonhuman primates, causing severe infections that are often fatal. The epidemics, in West and Central Africa, emphasize the urgent need to develop antiviral therapies. The Ebola virus large protein (L), which is the central protein for viral RNA replication/transcription, harbors a methyltransferase domain followed by a C-terminal domain of unknown function. We show that the C-terminal domain regulates the L protein methyltransferase activities and consequently participates in viral replication and escape of the host innate immunity.

DNA processing by the MOBH family relaxase TraI encoded within the gonococcal genetic island.

Relaxases of the MOBH family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOBH family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn2+ or Co2+ dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3′ of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site.

Crystal Structure of Aldehyde Dehydrogenase 16 Reveals Trans-Hierarchical Structural Similarity and a New Dimer

The aldehyde dehydrogenase (ALDH) superfamily is a vast group of enzymes that catalyze the NAD+-dependent oxidation of aldehydes to carboxylic acids. ALDH16 is perhaps the most enigmatic member of the superfamily, owing to its extra C-terminal domain of unknown function and the absence of the essential catalytic cysteine residue in certain non-bacterial ALDH16 sequences. Herein we report the first production of recombinant ALDH16, the first biochemical characterization of ALDH16, and the first crystal structure of ALDH16. Recombinant expression systems were generated for the bacterial ALDH16 from Loktanella sp. and human ALDH16A1. Four high-resolution crystal structures of Loktanella ALDH16 were determined. Loktanella ALDH16 is found to be a bona fide enzyme, exhibiting NAD+-binding, ALDH activity, and esterase activity. In contrast, human ALDH16A1 apparently lacks measurable aldehyde oxidation activity, suggesting that it is a pseudoenzyme, consistent with the absence of the catalytic Cys in its sequence. The fold of ALDH16 comprises three domains: NAD+-binding, catalytic, and C-terminal. The latter is unique to ALDH16 and features a Rossmann fold connected to a protruding β-flap. The tertiary structural interactions of the C-terminal domain mimic the quaternary structural interactions of the classic ALDH superfamily dimer, a phenomenon we call “trans-hierarchical structural similarity.” ALDH16 forms a unique dimer in solution, which mimics the classic ALDH superfamily dimer-of-dimer tetramer. Small-angle X-ray scattering shows that human ALDH16A1 has the same dimeric structure and fold as Loktanella ALDH16. We suggest that the Loktanella ALDH16 structure may be considered to be the archetype of the ALDH16 family.

PE_PGRS3 of Mycobacterium tuberculosis is specifically expressed at low phosphate concentration, and its arginine-rich C-terminal domain mediates adhesion and persistence in host tissues when expressed in Mycobacterium smegmatis.

PE_PGRSs of Mycobacterium tuberculosis (Mtb) represent a family of complex and peculiar proteins whose role and function remain elusive. In this study, we investigated PE_PGRS3 and PE_PGRS4, two highly homologous PE_PGRSs encoded by two contiguous genes in the Mtb genome. Using a gene-reporter system in Mycobacterium smegmatis (Ms) and transcriptional analysis in Mtb, we show that PE_PGRS3, but not PE_PGRS4, is specifically expressed under low phosphate concentrations. Interestingly, PE_PGRS3, but not PE_PGRS4, has a unique, arginine-rich C-terminal domain of unknown function. Heterologous expression of PE_PGRS3 in Ms was used to demonstrate cellular localisation of the protein on the mycobacterial surface, where it significantly affects net surface charge. Moreover, expression of full-length PE_PGRS3 enhanced adhesion of Ms to murine macrophages and human epithelial cells and improved bacterial persistence in spleen tissue following infection in mice. Expression of the PE_PGRS3 functional deletion mutant lacking the C-terminal domain in Ms did not enhance adhesion to host cells, showing a phenotype similar to the Ms parental strain. Interestingly, enhanced persistence of Ms expressing PE_PGRS3 did not correlate with increased concentrations of inflammatory cytokines. These results point to a critical role for the ≈ 80 amino acids long, arginine-rich C-terminal domain of PE_PGRS3 in tuberculosis pathogenesis.

Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) also interacts with WOX and KNOX proteins associated with wood formation in Populus trichocarpa.

DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) from snapdragon (Antirrhinum majus) is a MYB/SANT protein that interacts with related MYB/SANT proteins, RADIALIS and DIVARICATA, through its N-terminal MYB/SANT domain. In addition to the MYB/SANT domain, DRIF contains a C-terminal domain of unknown function (DUF3755). Here we describe novel protein-protein interactions involving a poplar (Populus trichocarpa) homolog of DRIF, PtrDRIF1. In addition to interacting with poplar homologs of RADIALIS (PtrRAD1) and DIVARICATA (PtrDIV4), PtrDRIF1 interacted with members of other families within the homeodomain-like superfamily, including PtrWOX13c, a WUSCHEL-RELATED HOMEOBOX protein, and PtrKNAT7, a KNOTTED1-LIKE HOMEOBOX protein. PtrRAD1 and PtrDIV4 interacted with the MYB/SANT-containing N-terminal portion of PtrDRIF1, whereas DUF3755 was both necessary and sufficient for interactions with PtrWOX13c and PtrKNAT7. Of the two MYB/SANT domains present in PtrDIV4, only the N-terminal MYB/SANT domain interacted with PtrDRIF1. GFP-PtrDRIF1 expressed alone or with PtrRAD1 localized to the cytoplasm, whereas co-expression of GFP-PtrDRIF1 with PtrDIV4, PtrWOX13c or PtrKNAT7 resulted in nuclear localization of GFP-PtrDRIF1. Modified yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments using PtrDRIF1 as a bridge protein revealed that PtrDRIF1 simultaneously interacted with PtrRAD1 and PtrWOX13c, but could not form a heterotrimeric complex when PtrDIV4 was substituted for PtrRAD1. Moreover, a Y2H competition assay indicated that PtrKNAT7 inhibits the interaction between PtrRAD1 and PtrDRIF1. The discovery of an additional protein-protein interaction domain in DRIF proteins, DUF3755, and its ability to form heterodimers and heterotrimers involving MYB/SANT and wood-associated homeodomain proteins, implicates DRIF proteins as mediators of a broader array of processes than previously reported.

Evidence that the C-terminal domain of a type B PutA protein contributes to aldehyde dehydrogenase activity and substrate channeling.

Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH–P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target–decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium.

Structural basis of substrate channeling in the PutA flavoenzyme

Proline utilization A (PutA) is a high-hanging fruit of X-ray crystallography. PutA is a membrane-associated bifunctional flavoenzyme that catalyzes the 4-electron oxidation of proline to glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. PutAs are challenging crystallography targets because of their long polypeptide chain length (1000-1300 residues) and multidomain architecture. In this talk, I will present new crystal structures and SAXS analysis of two PutAs. Seven high resolution crystal structures of a 1004-residue minimalist PutA were determined using Hg SIRAS phasing, and the oligomeric state and quaternary structure were determined with SAXS [1]. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å long tunnel that links the two active sites. Also, a novel mechanism-based inactivation strategy allowed the trapping of the elusive PutA-quinone complex in the crystalline state. These structures provide insight into the mechanism of substrate channeling and how the enzyme changes conformation during the catalytic cycle. I will conclude by describing the first structure of a new type of PutA that contains an additional C-terminal domain of unknown function (CTDUF) that is not present in the smaller minimalist enzyme [2]. This larger PutA reveals an unexpectedly different structural solution to the problem of sequestering the reaction intermediate.

Mutational Analyses Reveal Overall Topology and Functional Regions of NilB, a Bacterial Outer Membrane Protein Required for Host Association in a Model of Animal-Microbe Mutualism

The gammaproteobacterium Xenorhabdus nematophila is a mutualistic symbiont that colonizes the intestine of the nematode Steinernema carpocapsae. nilB (nematode intestine localization) is essential for X. nematophila colonization of nematodes and is predicted to encode an integral outer membrane beta-barrel protein, but evidence supporting this prediction has not been reported. The function of NilB is not known, but when expressed with two other factors encoded by nilA and nilC, it confers upon noncognate Xenorhabdus spp. the ability to colonize S. carpocapsae nematodes. We present evidence that NilB is a surface-exposed outer membrane protein whose expression is repressed by NilR and growth in nutrient-rich medium. Bioinformatic analyses reveal that NilB is the only characterized member of a family of proteins distinguished by N-terminal region tetratricopeptide repeats (TPR) and a conserved C-terminal domain of unknown function (DUF560). Members of this family occur in diverse bacteria and are prevalent in the genomes of mucosal pathogens. Insertion and deletion mutational analyses support a beta-barrel structure model with an N-terminal globular domain, 14 transmembrane strands, and seven extracellular surface loops and reveal critical roles for the globular domain and surface loop 6 in nematode colonization. Epifluorescence microscopy of these mutants demonstrates that NilB is necessary at early stages of colonization. These findings are an important step in understanding the function of NilB and, by extension, its homologs in mucosal pathogens.

A Mutant Defective in Sexual Development Produces Aseptate Ascogonia

The transition from the vegetative to the sexual cycle in filamentous ascomycetes is initiated with the formation of ascogonia. Here, we describe a novel type of sterile mutant from Sordaria macrospora with a defect in ascogonial septum formation. This mutant, named pro22, produces only small, defective protoperithecia and carries a point mutation in a gene encoding a protein that is highly conserved throughout eukaryotes. Sequence analyses revealed three putative transmembrane domains and a C-terminal domain of unknown function. Live-cell imaging showed that PRO22 is predominantly localized in the dynamic tubular and vesicular vacuolar network of the peripheral colony region close to growing hyphal tips and in ascogonia; it is absent from the large spherical vacuoles in the vegetative hyphae of the subperipheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar network, and the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.

Solution structure of the C-terminal DUF1000 domain of the human thioredoxin-like 1 protein

Solution structure of the C-terminal DUF1000 domain of the human thioredoxin-like 1 protein

Crystal structure of native cinnamomin isoform III and its comparison with other ribosome inactivating proteins

Crystal structure of native cinnamomin isoform III and its comparison with other ribosome inactivating proteins Arezki Azzi,1,2y Tao Wang,1y Dao-Wei Zhu, Yong-Shui Zou, Wang-Yi Liu, and Sheng-Xiang Lin* 1 Structural Biology Platform, Institute of Biochemistry and Cell Biology (IBCB), Shanghai Institutes of Biological Sciences (SIBS), Chinese Academy of Sciences, China 2 Laboratory of Oncology and Molecular Endocrinology, CHUL Research Center (CHUQ) and Laval University, Quebec, Canada

Expression of TMEM16 paralogs during murine embryogenesis

The TMEM16 protein family has recently been identified through several different experimental strategies including bioinformatic and microarray-based approaches. In mice and humans, there exist 10 paralogs with each containing eight putative transmembrane domains and a conserved C-terminal domain of unknown function. Mutation of at least one member of this family is associated with a human disorder, and several members of this gene family are overexpressed in different types of cancer. Despite their apparent relevance to normal development and disease, little is known about the expression of TMEM16 paralogs during embryonic development. Here, we provide a phylogenetic analysis of mouse and human TMEM16 paralogs and report the expression of Tmem16a, Tmem16b, Tmem16c, Tmem16f, Tmem16h, Tmem16j, and Tmem16k during murine embryogenesis with an emphasis on the respiratory, digestive, skeletal, and integumentary systems. These data should encourage investigations into the functions of TMEM16 paralogs in vertebrate development.

The txl1+ gene from Schizosaccharomyces pombe encodes a new thioredoxin‐like 1 protein that participates in the antioxidant defence against tert‐butyl hydroperoxide

Yeasts are equipped with several putative single-domain thioredoxins located in different subcellular compartments. However, additional proteins containing thioredoxin domains are also encoded by the yeast genomes as described for mammals and other eukaryotic organisms. We report here the characterization of the fission yeast orthologue thioredoxin-like 1 (txl1(+)), which has been previously identified in mammals. Similarly to the human protein, the fission yeast Txl1 is a two-domain protein comprising an N-terminal thioredoxin-like domain and a C-terminal domain of unknown function. Many other yeasts and fungi species contain homologues of txl1(+); however, there is no evidence of txl1(+) orthologues in either Saccharomyces cerevisiae or plants. Txl1 is found in both the nucleus and the cytoplasm of Schizosaccharomyces pombe cells and exhibits a strong reducing activity coupled to thioredoxin reductase. In humans, TXL1 expression is induced by glucose deprivation and overexpression of TXL1 confers resistance against this stress. In contrast, a Sz. pombe Deltatxl1 mutant was not affected in the response against glucose starvation but the Deltatxl1 mutant strain showed a clear hypersensitivity to alkyl hydroperoxide. The mRNA levels of txl1(+) in a h20 strain did not change in response to any oxidative insult (hydrogen peroxide or alkyl hydroperoxide) and the overexpression of an integrated copy of the wild-type txl1(+) gene did not confer a significant increased resistance against alkyl hydroperoxide. Overall, these results indicate that the Txl1 role in the cellular detoxification of alkyl hydroperoxide is exerted through a constitutive transcription of txl1(+).