Caspase-3 Regulates YAP-Dependent Cell Proliferation and Organ Size.

Apoptosis culminates in the activation of caspase-3, which plays an important role in implementing the cell death program. Here, we reveal a non-apoptotic role of caspase-3 as a key regulator of cell proliferation and organ size. Caspase-3 is specifically activated in the proliferating cells of the sebaceous gland, but does not instruct cell elimination. Deletion or chemical inhibition of caspase-3 diminishes cell proliferation, decreases cell number and reduces sebaceous gland size in vivo. Exploring the underlying mechanism, we demonstrate that α-catenin is cleaved by caspase-3, thus facilitating the activation and nuclear translocation of yes-associated protein (YAP), a vital regulator of organ size. Accordingly, activation of caspase-3 leads to YAP-dependent organ size augmentation. Finally, we show that X-linked inhibitor of apoptosis protein (XIAP) serves as an endogenous feedback antagonist for the caspase-3/YAP signaling module. Taken together, we report here a molecular mechanism wherein the apoptotic machinery is refocused to regulate cell proliferation and orchestrate organ size.

Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

Porphyromonas gingivalis induction of TLR2 association with Vinculin enables PI3K activation and immune evasion.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that thrives in the inflamed environment of the gingival crevice, and is strongly associated with periodontal disease. The host response to P. gingivalis requires TLR2, however P. gingivalis benefits from TLR2-driven signaling via activation of PI3K. We studied TLR2 protein-protein interactions induced in response to P. gingivalis, and identified an interaction between TLR2 and the cytoskeletal protein vinculin (VCL), confirmed using a split-ubiquitin system. Computational modeling predicted critical TLR2 residues governing the physical association with VCL, and mutagenesis of interface residues W684 and F719, abrogated the TLR2-VCL interaction. In macrophages, VCL knock-down led to increased cytokine production, and enhanced PI3K signaling in response to P. gingivalis infection, effects that correlated with increased intracellular bacterial survival. Mechanistically, VCL suppressed TLR2 activation of PI3K by associating with its substrate PIP2. P. gingivalis induction of TLR2-VCL led to PIP2 release from VCL, enabling PI3K activation via TLR2. These results highlight the complexity of TLR signaling, and the importance of discovering protein-protein interactions that contribute to the outcome of infection.

Coronaviral PLpro proteases and the immunomodulatory roles of conjugated versus free Interferon Stimulated Gene product-15 (ISG15).

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits.

Analysis of the humoral and cellular response after the third COVID-19 vaccination in patients with autoimmune hepatitis.

BACKGROUND & AIMS: To explore the humoral and T-cell response to the third COVID-19 vaccination in autoimmune hepatitis (AIH). METHODS: Anti-SARS-CoV-2 antibody titers were prospectively determined in 81 AIH patients and 53 healthy age- and sex-matched controls >7 days (median 35) after the first COVID-19 booster vaccination. The spike-specific T-cell response was assessed using an activation-induced marker assay (AIM) in a subset of patients. RESULTS: Median antibody levels were significantly lower in AIH compared to controls (10 908 vs. 25 000 AU/ml, p < .001), especially in AIH patients treated with MMF (N = 14, 4542 AU/ml, p = .004) or steroids (N = 27, 7326 AU/ml, p = .020). Also, 48% of AIH patients had antibody titers below the 10% percentile of the healthy controls (9194 AU/ml, p < .001). AIH patients had a high risk of failing to develop a spike-specific T-cell response (15/34 (44%) vs. 2/16 (12%), p = .05) and showed overall lower frequencies of spike-specific CD4 + T cells (median: 0.074% vs 0.283; p = .01) after the booster vaccination compared to healthy individuals. In 34/81 patients, antibody titers before and after booster vaccination were available. In this subgroup, all patients but especially those without detectable/low antibodies titers (<100 AU/ml) after the second vaccination (N = 11/34) showed a strong, 148-fold increase. CONCLUSION: A third COVID-19 vaccination efficiently boosts antibody levels and T-cell responses in AIH patients and even seroconversion in patients with the absent immune response after two vaccinations, but to a lower level compared to controls. Therefore, we suggest routinely assessing antibody levels in AIH patients and offering additional booster vaccinations to those with suboptimal responses.

Autosomal dominant non-syndromic hearing loss maps to DFNA33 (13q34) and co-segregates with splice and frameshift variants in ATP11A, a phospholipid flippase gene.

Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3' UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.

Ferritin is secreted via 2 distinct nonclassical vesicular pathways.

Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum-Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body-exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.

Correction: Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter.

[This corrects the article DOI: 10.1371/journal.pcbi.1006503.].

The Arabidopsis chloroplast RNase J displays both exo- and robust endonucleolytic activities.

KEY MESSAGE: Arabidopsis chloroplast RNase J displaces both exo- and endo-ribonucleolytic activities and contains a unique GT-1 DNA binding domain. Control of chloroplast gene expression is predominantly at the post-transcriptional level via the coordinated action of nuclear encoded ribonucleases and RNA-binding proteins. The 5' end maturation of mRNAs ascribed to the combined action of 5'→3' exoribonuclease and gene-specific RNA-binding proteins of the pentatricopeptide repeat family and others that impede the progression of this nuclease. The exo- and endoribonuclease RNase J, the only prokaryotic 5'→3' ribonuclease that is commonly present in bacteria, Archaea, as well as in the chloroplasts of higher plants and green algae, has been implicated in this process. Interestingly, in addition to the metalo-ß-lactamase and ß-CASP domains, RNase J of plants contains a conserved GT-1 domain that was previously characterized in transcription factors that function in light and stress responding genes. Here, we show that the Arabidopsis RNase J (AtRNase J), when analyzed in vitro with synthetic RNAs, displays both 5'→3' exonucleolytic activity, as well as robust endonucleolytic activity as compared to its bacterial homolog RNase J1 of Bacillus subtilis. AtRNase J degraded single-stranded RNA and DNA molecules but displays limited activity on double stranded RNA. The addition of three guanosines at the 5' end of the substrate significantly inhibited the degradation activity, indicating that the sequence and structure of the RNA substrate modulate the ribonucleolytic activity. Mutation of three amino acid in the catalytic reaction center significantly inhibited both the endonucleolytic and exonucleolytic degradation activities, while deletion of the carboxyl GT-1 domain that is unique to the plant RNAse J proteins, had a little or no significant effect. The robust endonucleolytic activity of AtRNase J suggests its involvement in the processing and degradation of RNA in the chloroplast.

Liver infiltrating T cells regulate bile acid metabolism in experimental cholangitis.

BACKGROUND & AIMS: T cells are central mediators of liver inflammation and represent potential treatment targets in cholestatic liver disease. Whereas emerging evidence shows that bile acids (BAs) affect T cell function, the role of T cells for the regulation of BA metabolism is unknown. In order to understand this interplay, we investigated the influence of T cells on BA metabolism in a novel mouse model of cholangitis. METHODS: Mdr2-/- mice were crossed with transgenic K14-OVAp mice, which express an MHC class I restricted ovalbumin peptide on biliary epithelial cells (Mdr2-/-xK14-OVAp). T cell-mediated cholangitis was induced by the adoptive transfer of antigen-specific CD8+ T cells. BA levels were quantified using a targeted liquid chromatography-mass spectrometry-based approach. RESULTS: T cell-induced cholangitis resulted in reduced levels of unconjugated BAs in the liver and significantly increased serum and hepatic levels of conjugated BAs. Genes responsible for BA synthesis and uptake were downregulated and expression of the bile salt export pump was increased. The transferred antigen-specific CD8+ T cells alone were able to induce these changes, as demonstrated using Mdr2-/-xK14-OVAp recipient mice on the Rag1-/- background. Mechanistically, we showed by depletion experiments that alterations in BA metabolism were partly mediated by the proinflammatory cytokines TNF and IFN-γ in an FXR-dependent manner, a process that in vitro required cell contact between T cells and hepatocytes. CONCLUSION: Whereas it is known that BA metabolism is dysregulated in sepsis and related conditions, we have shown that T cells are able to control the synthesis and metabolism of BAs, a process which depends on TNF and IFN-γ. Understanding the effect of lymphocytes on BA metabolism will help in the design of combined treatment strategies for cholestatic liver diseases. LAY SUMMARY: Dysregulation of bile acid metabolism and T cells can contribute to the development of cholangiopathies. Before targeting T cells for the treatment of cholangiopathies, it should be determined whether they exert protective effects on bile acid metabolism. Herein, we demonstrate that T cell-induced cholangitis resulted in decreased levels of harmful unconjugated bile acids. T cells were able to directly control synthesis and metabolism of bile acids, a process which was dependent on the proinflammatory cytokines TNF and IFN-γ. Understanding the effect of lymphocytes on bile acid metabolism will help in the design of combined treatment strategies for cholestatic liver diseases.

Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter.

Multiscale modeling provides a very powerful means of studying complex biological systems. An important component of this strategy involves coarse-grained (CG) simplifications of regions of the system, which allow effective exploration of complex systems. Here we studied aspects of CG modeling of the human zinc transporter ZnT2. Zinc is an essential trace element with 10% of the proteins in the human proteome capable of zinc binding. Thus, zinc deficiency or impairment of zinc homeostasis disrupt key cellular functions. Mammalian zinc transport proceeds via two transporter families: ZnT and ZIP; however, little is known about the zinc permeation pathway through these transporters. As a step towards this end, we herein undertook comprehensive computational analyses employing multiscale techniques, focusing on the human zinc transporter ZnT2 and its bacterial homologue, YiiP. Energy calculations revealed a favorable pathway for zinc translocation via alternating access. We then identified key residues presumably involved in the passage of zinc ions through ZnT2 and YiiP, and functionally validated their role in zinc transport using site-directed mutagenesis of ZnT2 residues. Finally, we use a CG Monte Carlo simulation approach to sample the transition between the inward-facing and the outward-facing states. We present our structural models of the inward- and outward-facing conformations of ZnT2 as a blueprint prototype of the transporter conformations, including the putative permeation pathway and participating residues. The insights gained from this study may facilitate the delineation of the pathways of other zinc transporters, laying the foundations for the molecular basis underlying ion permeation. This may possibly facilitate the development of therapeutic interventions in pathological states associated with zinc deficiency and other disorders based on loss-of-function mutations in solute carriers.

Establishing the role of PLVAP in protein-losing enteropathy: a homozygous missense variant leads to an attenuated phenotype.

BACKGROUND: Intestinal integrity is essential for proper nutrient absorption and tissue homeostasis, with damage leading to enteric protein loss, that is, protein-losing enteropathy (PLE). Recently, homozygous nonsense variants in the plasmalemma vesicle-associated protein gene (PLVAP) were reported in two patients with severe congenital PLE. PLVAP is the building block of endothelial cell (EC) fenestral diaphragms; its importance in barrier function is supported by mouse models of Plvap deficiency. OBJECTIVE: To genetically diagnose two first-degree cousins once removed, who presented with PLE at ages 22 and 2.5 years. METHODS: Family-based whole exome sequencing was performed based on an autosomal recessive inheritance model. In silico analyses were used to predict variant impact on protein structure and function. RESULTS: We identified a rare homozygous variant (NM_031310.2:c.101T>C;p.Leu34Pro) in PLVAP, which co-segregated with the disease. Leu34 is predicted to be located in a highly conserved, hydrophobic, α-helical region within the protein's transmembrane domain, suggesting Leu34Pro is likely to disrupt protein function and/or structure. Electron microscopy and PLVAP immunohistochemistry demonstrated apparently normal diaphragm morphology, predicted to be functionally affected. CONCLUSIONS: Biallelic missense variants in PLVAP can cause an attenuated form of the PLE and hypertriglyceridaemia syndrome. Our findings support the role of PLVAP in the pathophysiology of PLE, expand the phenotypic and mutation spectrums and underscore PLVAP's importance in EC barrier function in the gut.

The Peptide Repertoire of HLA-B27 may include Ligands with Lysine at P2 Anchor Position.

The HLA-B*27 peptidome has drawn significant attention due to the genetic association between some of the HLA-B*27 alleles and the inflammatory rheumatic disease ankylosing spondylitis (AS), for which a comprehensive biological explanation is still lacking. This study aims to expand the known limits of the HLA-B*27 peptidome to facilitate selection and testing of new peptides, possibly involved in the disease. The HLA peptidomes of HeLa and C1R cell lines stably transfected with the AS-associated HLA-B*27:05 allele, the nonassociated HLA-B*27:09 allele, or their cysteine 67 to serine mutants (C67S), are analyzed on a very large scale. In addition, the peptidomes of HLA-B*27:05 and HLA-B*27:05-C67S are analyzed from the spleens of rats transgenic for these alleles. The results indicate that C67S mutation increases the percentage of peptides with glutamine or lysine at their P2 position (P2-Lys), in both HLA-B*27:05 and HLA-B*27:09. Furthermore, a small fraction of HLA-B*27 peptides contains lysine at their second position (P2), in addition to the more commonly found peptides with arginine (P2-Arg) or the less common glutamine (P2-Gln) located at this anchor position. Overall these data indicate that peptides with P2-Lys should be considered as real ligands of HLA-B*27 molecules and taken into account while looking for putative peptides implicated in the AS.

Molecular Basis of Transient Neonatal Zinc Deficiency: NOVEL ZnT2 MUTATIONS DISRUPTING ZINC BINDING AND PERMEATION.

A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations. Here we characterized the impact of three novel heterozygous ZnT2 mutations G280R, T312M, and E355Q, which cause TNZD in exclusively breastfed infants of Japanese mothers. We used the bimolecular fluorescence complementation (BiFC) assay to provide direct visual evidence for the in situ dimerization of these ZnT2 mutants, and to explore their subcellular localization. Moreover, using three complementary functional assays, zinc accumulation using BiFC-Zinquin and Zinpyr-1 fluorescence as well as zinc toxicity assay, we determined the impact of these ZnT2 mutations on vesicular zinc accumulation. Although all three mutants formed homodimers with the wild type (WT) ZnT2 and retained substantial vesicular localization, as well as vesicular zinc accumulation, they had no dominant-negative effect over the WT ZnT2. Furthermore, using advanced bioinformatics, structural modeling, and site-directed mutagenesis we found that these mutations localized at key residues, which play an important physiological role in zinc coordination (G280R and E355Q) and zinc permeation (T312M). Collectively, our findings establish that some heterozygous loss of function ZnT2 mutations disrupt zinc binding and zinc permeation, thereby suggesting a haploinsufficiency state for the unaffected WT ZnT2 allele in TNZD pathogenesis. These results highlight the burning need for the development of a suitable genetic screen for the early diagnosis of TNZD to prevent morbidity.

Dysfunction of hepatic regulatory T cells in experimental sclerosing cholangitis is related to IL-12 signaling.

BACKGROUND & AIMS: Reduced numbers of regulatory T cells (Treg) have been reported in patients with primary sclerosing cholangitis (PSC); therefore, Treg expansion might serve as a therapeutic approach. Here, we explored whether treatment with IL-2/IL-2 monoclonal antibody complex (IL-2/IL-2Ab complex) could provide in vivo Treg expansion and treatment of experimental sclerosing cholangitis. METHODS: Treg were expanded by repeated injection of IL-2/IL-2Ab complex in mouse models of cholangitis (Mdr2-/-, DDC) or colitis (dextran sulfate sodium [DSS]) as control. In vitro suppressive capacity and gene expression were analyzed in isolated hepatic and splenic Treg. RESULTS: In vivo expansion resulted in a 5-fold increase in hepatic Treg, which localized within the inflamed portal tracts. However, although Treg expansion was associated with reduced pro-inflammatory IL-17 and increased anti-inflammatory IL-10 production by hepatic lymphocytes, the severity of cholangitis was not reduced. In contrast, DSS-induced colitis could be improved by Treg expansion, suggesting a selectively reduced functionality of intrahepatic Treg. Indeed, hepatic Treg manifested reduced Foxp3 expression and reduced suppressive capacity compared to splenic Treg. Hepatic Treg dysfunction could be linked to increased IL-12 signaling due to an upregulation of the IL-12 receptor. Accordingly, IL-12 receptor beta 2 knockout mice (IL-12rb2-/-) were able to maintain hepatic Treg functionality. CONCLUSIONS: Hepatic Treg expanded in vivo failed to improve the course of cholangitis, which was related to the effects of hepatic IL-12 on Treg. Therefore, neutralization of IL-12 should be considered as part of treatment strategies targeting Treg in sclerosing cholangitis. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is associated with a paucity of regulatory T cells (Treg) that have a particular ability to control immune responses; therefore, in vivo expansion of Treg might serve as a treatment of cholangitis. However, in a mouse model of PSC, we show that Treg enrichment in the liver was not sufficient to provide effective control of cholangitis, as the suppressive functionality of hepatic Treg was significantly limited by IL-12 signals. Thus, neutralization of IL-12 should be considered as part of treatment strategies to improve the efficacy of Treg-based treatments for liver diseases. Data accession number: GSE 87898.

In situ dimerization of multiple wild type and mutant zinc transporters in live cells using bimolecular fluorescence complementation.

Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells.

Photosystem I gene cassettes are present in marine virus genomes.

Cyanobacteria of the Synechococcus and Prochlorococcus genera are important contributors to photosynthetic productivity in the open oceans. Recently, core photosystem II (PSII) genes were identified in cyanophages and proposed to function in photosynthesis and in increasing viral fitness by supplementing the host production of these proteins. Here we show evidence for the presence of photosystem I (PSI) genes in the genomes of viruses that infect these marine cyanobacteria, using pre-existing metagenomic data from the global ocean sampling expedition as well as from viral biomes. The seven cyanobacterial core PSI genes identified in this study, psaA, B, C, D, E, K and a unique J and F fusion, form a cluster in cyanophage genomes, suggestive of selection for a distinct function in the virus life cycle. The existence of this PSI cluster was confirmed with overlapping and long polymerase chain reaction on environmental DNA from the Northern Line Islands. Potentially, the seven proteins encoded by the viral genes are sufficient to form an intact monomeric PSI complex. Projection of viral predicted peptides on the cyanobacterial PSI crystal structure suggested that the viral-PSI components might provide a unique way of funnelling reducing power from respiratory and other electron transfer chains to the PSI.

Conformational transitions in human translin enable nucleic acid binding.

Translin is a highly conserved RNA- and DNA-binding protein that plays essential roles in eukaryotic cells. Human translin functions as an octamer, but in the octameric crystallographic structure, the residues responsible for nucleic acid binding are not accessible. Moreover, electron microscopy data reveal very different octameric configurations. Consequently, the functional assembly and the mechanism of nucleic acid binding by the protein remain unclear. Here, we present an integrative study combining small-angle X-ray scattering (SAXS), site-directed mutagenesis, biochemical analysis and computational techniques to address these questions. Our data indicate a significant conformational heterogeneity for translin in solution, formed by a lesser-populated compact octameric state resembling the previously solved X-ray structure, and a highly populated open octameric state that had not been previously identified. On the other hand, our SAXS data and computational analyses of translin in complex with the RNA oligonucleotide (GU)12 show that the internal cavity found in the octameric assemblies can accommodate different nucleic acid conformations. According to this model, the nucleic acid binding residues become accessible for binding, which facilitates the entrance of the nucleic acids into the cavity. Our data thus provide a structural basis for the functions that translin performs in RNA metabolism and transport.

The hnRNP F/H homologue of Trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability.

Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation. In this study, we performed functional analysis of the Trypanosoma brucei heterogeneous nuclear ribonucleoproteins (hnRNP) F/H homologue, a protein known to regulate alternative splicing in metazoa. The hnRNP F/H is highly expressed in the bloodstream form of the parasite, but is also functional in the procyclic form. Transcriptome analyses of RNAi-silenced cells were used to deduce the RNA motif recognized by this protein. A purine rich motif, AAGAA, was enriched in both the regulatory regions flanking the 3' splice site and poly (A) sites of the regulated genes. The motif was further validated using mini-genes carrying wild-type and mutated sequences in the 3' and 5' UTRs, demonstrating the role of hnRNP F/H in mRNA stability and splicing. Biochemical studies confirmed the binding of the protein to this proposed site. The differential expression of the protein and its inverse effects on mRNA level in the two lifecycle stages demonstrate the role of hnRNP F/H in developmental regulation.