Retinoid-related orphan receptor γ (RORγ) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis

To identify the physiological functions of the retinoid-related orphan receptor γ (RORγ), a member of the nuclear receptor superfamily, mice deficient in RORγ function were generated by targeted disruption. RORγ−/− mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that RORγ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of RORγ−/− mice contains 74.4% ± 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of RORγ−/− mice. The latter was supported by the observed increase in annexin V-positive cells. RORγ−/− thymocytes placed in culture exhibit a dramatic increase in the rate of “spontaneous” apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORγ−/− mice. Our observations indicate that RORγ is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which RORγ negatively controls apoptosis in thymocytes.

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte–antigen-presenting cell interface

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav−/− mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav+/− mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav−/− mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: Clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant

A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52/2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription–PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.

RAD50 function is essential for telomere maintenance in Arabidopsis

We have identified and characterized an Arabidopsis thaliana rad50 mutant plant containing a T-DNA insertion in the AtRAD50 gene and showing both meiotic and DNA repair defects. We report here that rad50/rad50 mutant cells show a progressive shortening of telomeric DNA relative to heterozygous rad50/RAD50 controls and that the mutant cell population rapidly enters a crisis, with the majority of the cells dying. Surviving rad50 mutant cells have longer telomeres than wild-type cells, indicating the existence in plants of an alternative RAD50-independent mechanism for telomere maintenance. These results report the role of a protein essential for double-strand break repair in telomere maintenance in higher eukaryotes.

Splicing of constitutive upstream introns is essential for the recognition of intra-exonic suboptimal splice sites in the thrombopoietin gene

The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon–intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.

The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties

Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d1-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d1-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 Å from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd1 nitrite reductase from P. aeruginosa.

A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-κB activity in astrocytes

The neural cell adhesion molecule (N-CAM) is expressed on the surface of astrocytes, where its homophilic binding leads to the activation of the transcription factor NF-κB. Transfection of astrocytes with a construct encompassing the transmembrane region and the cytoplasmic domain of N-CAM (designated Tm-Cyto, amino acids 685–839 in the full-length molecule) inhibited this activation up to 40%, and inhibited N-CAM-induced translocation of NF-κB to the nucleus. N-CAM also activated NF-κB in astrocytes from N-CAM knockout mice, presumably through binding to a heterophile. This activation, however, was not blocked by Tm-Cyto expression, indicating that the inhibitory effect of the Tm-Cyto construct is specific for cell surface N-CAM. Deletions and point mutations of the cytoplasmic portion of the Tm-Cyto construct indicated that the region between amino acids 780 and 800 were essential for inhibitory activity. This region contains four threonines (788, 793, 794, and 797). Mutation to alanine of T788, T794, or T797, but not T793, abolished inhibitory activity, as did mutation of T788 or T797 to aspartic acid. A Tm-Cyto construct with T794 mutated to aspartic acid retained inhibitory activity but did not itself induce a constitutive NF-κB response. This result suggests that phosphorylation of T794 may be necessary but is not the triggering event. Overall, these findings define a short segment of the N-CAM cytoplasmic domain that is critical for N-CAM-induced activation of NF-κB and may be important in other N-CAM-mediated signaling.

KAT1 is not essential for stomatal opening

It is generally accepted that K+ uptake into guard cells via inward-rectifying K+ channels is required for stomatal opening. To test whether the guard cell K+ channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1∷En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K+ uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription–PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K+ channels AKT1, AKT2/3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inward-rectifying K+ currents across the plasma membrane of both wild-type and KAT1∷En-1 plants. This study demonstrates that multiple K+ channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1

We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met. It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif. The glutathione S-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376–405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380–408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction. The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48. These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein–protein interactions may not be necessary for this interaction of chHAT-1.

Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Bα Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Bα subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Bα subunit binding without greatly affecting methylation, indicating that Bα subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Bα subunit but not the amount that could be coimmunoprecipitated with Aα subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Bα subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Bα subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.

A transcriptional corepressor of Stat1 with an essential LXXLL signature motif

Interferon (IFN) treatment induces tyrosine phosphorylation and nuclear translocation of Stat1 (signal transducer and activator of transcription) to activate or repress transcription. We report here that a member of the protein inhibitor of activated STAT family, PIASy, is a transcriptional corepressor of Stat1. IFN treatment triggers the in vivo interaction of Stat1 with PIASy, which represses Stat1-mediated gene activation without blocking the DNA binding activity of Stat1. An LXXLL coregulator signature motif located near the NH2 terminus of PIASy, although not involved in the PIASy–Stat1 interaction, is required for the transrepression activity of PIASy. Our studies identify PIASy as a transcriptional corepressor of Stat1 and suggest that different PIAS proteins may repress STAT-mediated gene activation through distinct mechanisms.

An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids

Hepatic hydroxylation is an essential step in the metabolism and excretion of bile acids and is necessary to avoid pathologic conditions such as cholestasis and liver damage. In this report, we demonstrate that the human xenobiotic receptor SXR (steroid and xenobiotic receptor) and its rodent homolog PXR (pregnane X receptor) serve as functional bile acid receptors in both cultured cells and animals. In particular, the secondary bile acid derivative lithocholic acid (LCA) is highly hepatotoxic and, as we show here, a metabolic substrate for CYP3A hydroxylation. By using combinations of knockout and transgenic animals, we show that activation of SXR/PXR is necessary and sufficient to both induce CYP3A enzymes and confer resistance to toxicity by LCA, as well as other xenotoxicants such as tribromoethanol and zoxazolamine. Therefore, we establish SXR and PXR as bile acid receptors and a role for the xenobiotic response in the detoxification of bile acids.

Profilin I is essential for cell survival and cell division in early mouse development

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1ko) allele in mice. Homozygous pfn1ko/ko mice are not viable. Pfn1ko/ko embryos died as early as the two-cell stage, and no pfn1ko/ko blastocysts were detectable. Adult pfn1ko/wt mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1ko/wt embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.