Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3

Maturation of organelles in the endolysosomal pathway requires exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the guanine nucleotide exchange factor activity of the Mon1-Ccz1 heterodimer for Ypt7. Here we investigate vacuole binding and recycling of Mon1-Ccz1. We find that Mon1-Ccz1 is absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34, and the lipid phosphatidylinositol 3-phosphate (PI3P). Interaction of Mon1-Ccz1 with wild-type vacuoles requires PI3P, as shown in competition experiments. We also find that Mon1 is released from vacuoles during the fusion reaction and its release requires its phosphorylation by the type 1 casein kinase Yck3. In contrast, Mon1 is retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites are mutated. Phosphorylation and release of Mon1 is restored with addition of recombinant Yck3. Together the results show that Mon1 is recruited to endosomes and vacuoles by PI3P and, likely after activating Ypt7, is phosphorylated and released from vacuoles for recycling.     

Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics

Introduction

Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood.

Methods

We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy.

Results

We documented evidence for four major phenotypes of RA synovium – lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004).

Conclusions

These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01119859","term_id":"NCT01119859"}}NCT01119859     

Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana: is it caused by genetic variation in the STN kinases?

Reversible phosphorylation of photosystem II (PSII) proteins is an important regulatory mechanism that can protect plants from changes in ambient light intensity and quality. We hypothesized that there is natural variation in this process in Arabidopsis (Arabidopsis thaliana), and that this results from genetic variation in the STN7 and STN8 kinase genes. To test this, Arabidopsis accessions of diverse geographical origins were exposed to two light regimes, and the levels of phospho-D1 and phospho-light harvesting complex II (LHCII) proteins were quantified by western blotting with anti-phosphothreonine antibodies. Accessions were classified as having high, moderate or low phosphorylation relative to Col-0. This variation could not be explained by the abundance of the substrates in thylakoid membranes. In genotypes with atrazine-resistant forms of the D1 protein, low D1 and LHCII protein phosphorylation was observed, which may be due to low PSII efficiency, resulting in reduced activation of the STN kinases. In the remaining genotypes, phospho-D1 levels correlated with STN8 protein abundance in high-light conditions. In growth light, D1 and LHCII phosphorylation correlated with longitude and in the case of LHCII phosphorylation also with temperature variability. This suggests a possible role of natural variation in PSII protein phosphorylation in the adaptation of Arabidopsis to diverse environments.     

Plasma Levels of Nitrite and Nitrate in Early and Recent Classes of Fish

The stable metabolite of nitric oxide in plasma is NO_x, the sum of nitrite plus nitrate. Measures of plasma NO_x may provide information about the nitric oxide tonus of the entire endothelium including capillary microvessels. Although data are available for mammalian species, plasma NO_x measurements in early vertebrate species are scarce. The purpose of this study was to test the hypothesis that plasma NO_x would be similar to the NO_x in the water environment for fish in early classes (Agnatha and Chondrichthye) and would exceed water NO_x levels in the known nitrite-sensitive fish (Osteichthye). Plasma samples were obtained from 18 species of adult fish (n = 167) and from their housing or natural water environment. NO_x was measured by using chemiluminescence. Plasma NO_x was detected in all species and ranged from 0.5 nmol/ml (skate) to 453.9 nmol/ml (shortnose gar). Average plasma NO_x was significantly higher in sea lamprey than in Atlantic hagfish whereas that of little skate was 3-fold lower than in spiny dogfish shark. Plasma NO_x differed significantly among early bony fish (paddlefish, pallid sturgeon, gar) yet was similar among modern bony fish, with the exception of rainbow trout. Plasma NO_x reflected water NO_x in only 2 species (hagfish and shark), and levels did not coincide with nitrite sensitivity. This study provides an expanded comparative view of plasma NO_x levels across 3 groups of early fish. The data obtained suggest a nitric oxide system in early and modern fish.Abbreviations: NOS, nitric oxide synthaseNitric oxide is generated from oxygen and L-arginine by nitric oxide synthase (NOS), an enzyme with 3 isoforms: constitutive (or endothelial cell), neuronal, and inducible. In mammals, nitric oxide is an important signaling molecule that is responsible for functions in the cardiovascular, nervous, and immune systems.¹⁹ The role that nitric oxide plays as a vasodilator molecule in the peripheral circulation is of particular importance because it serves to regulate vascular tone and total peripheral resistance.⁹Comparative studies focused on nitric oxide provide valuable information about the conserved nature of this ubiquitous molecule. Staining for NADPH-diaphorase (an enzyme equivalent to NOS) or testing for reactivity of vessels isolated from the species of interest have been used to investigate the phylogenetic roots of NOS. Insects^13,20 as well as marine invertebrates, including the horseshoe crab with its copper-containing erythrocytes,²⁶ display NOS activity and produce measurable amounts of nitric oxide. Nitric oxide appears to be responsible for a wide variety of physiology including immune function, growth, development, and neural responses.Nitric oxide is released continuously into surrounding tissues as well as into circulation. From biochemistry studies nitric oxide apparently interacts with platelets and leukocytes²⁸ as well as with hemoglobin inside red blood cells, albeit more slowly than had been thought.³⁴ In addition to their interactions with cells, nitric oxide molecules bind to various proteins in plasma, including albumin, the most abundant protein in circulation.^28,29In vivo, nitric oxide also may exist in its unbound form.²⁷ The region along the vascular wall that remains free of erythrocytes has been suggested as 1 location of unbound nitric oxide with a biologic lifetime in the range of 100 to 500 s.⁶ This length of time allows nitric oxide to affect vessels downstream from its release point, thus performing a hormone-like function.²⁸The stable metabolite of nitric oxide in the plasma portion of blood is referred to as NO_x and is the sum of the oxidative products of nitric oxide, nitrite plus nitrate. Measures of plasma NO_x provide information about chronic basal NOS activity for the entire endothelium including capillary microvessels (accounting for the largest surface area of endothelial cells). Although nitrite appears to reflect acute changes in endothelial cell NOS activity in humans,¹⁴ reports indicate uniformity in both nitrite and NO_x levels across a range of mammalian species,^12,35 likely reflecting similarities in chronic basal NOS activity, that is, nitric oxide tonus.Few studies report measures of NO_x in the plasma of early vertebrate species. Previous work indicated differences in NO_x levels of 8 mammalian species compared with the other vertebrates that were tested.³⁵ One observation from that study was that NO_x was higher and more variable in plasma sampled from fish in the classes Agnatha, Chondrichthye, and Osteichthye. In the present study, the observations were expanded to include plasma NO_x levels for a range of fishes sampled from 3 groups of fish with a spectrum of nitrite sensitivity and from a variety of natural and research housing habitats. Water NO_x levels were measured also. Our hypothesis was that levels of NO_x circulating in plasma would be similar to NO_x in the water environment for fish in the early classes (Agnatha and Chondrichthye) and above water NO_x levels only in the nitrite-sensitive fish of the Osteichthye class. Average NO_x for lamprey, skate, and trout have been published previously.³⁵     

Unexpected diversity of bioluminescence in planktonic worms

The vast majority of pelagic bioluminescent organisms emit a blue light with emission maxima (λ_max) ranging from 450 to 490 nm. Among the known outliers, the tomopterids (Annelida: Polychaeta) are usually described as yellow‐emitters (λ_max = 565–570 nm) for which bioluminescence functions as a specific recognition signal. Here, we report the first data regarding the colours emitted by four different tomopterid species, Tomopteris pacifica, T. carpenteri, T. septentrionalis and T. planktonis. Surprisingly, T. planktonis is a blue‐emitter (λ_max = 450 nm). Our pharmacological results on T. planktonis support cholinergic control, as recently demonstrated in the yellow‐emitter, T. helgolandica. Moreover, as revealed by epifluorescence microscopy, the light seems to be produced in both species from the same yellow‐pigmented parapodial glands. Despite these similarities, tomopterids express an unexpected diversity of bioluminescent colour patterns. This leads us to reassess the ecological value of bioluminescence within this group.     

Regulatory responses in contact sensitivity: afferent suppressor T cells inhibit the activation of efferent suppressor T cells.

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.     

A monoclonal antibody that recognizes an Ly-6-linked antigen inhibits the generation of functionally active T cell subsets

A monoclonal antibody (mAb) generated against the chemically-induced BALB/c Meth A sarcoma, designated HD42, reacts in cytotoxic tests with Meth A as well as with BALB/c peripheral lymph node cells and mitogen-activated spleen cells. The antigen was detected by FACS analysis on BALB/c spleen and lymph node cells, and by absorption assays on all normal lymphoid cells of BALB/c but not B6 mice. The expression of the antigen was not found on normal adult lung fibroblasts, on brain, nor on an extensive panel of tumors of BALB/c and B6 origin. Because the strain distribution of the antigen is reciprocal to that of Ly-6.2 and is not expressed in congenic C3H.Ly-6b mice, we have tentatively defined it as Ly-6.1 and referred to the mAb as alpha-Ly-6.1. The presence of alpha-Ly-6.1 abrogates both the Con A-induced and the IL 2-dependent proliferative response of normal T cells, whereas the response of normal B cells to LPS remains unaffected. alpha-Ly-6.1 is a potent suppressor of the primary in vitro plaque-forming cell (PFC) response to SRBC. Pretreatment of normal splenic T cells with alpha-Ly-6.1 and complement had no effect on the ability of these cells to generate in vitro either T helper cells (TH) or T suppressor cells (TS) to SRBC. However, addition of antibody in the absence of complement during the generation of TH or TS, or posttreatment of these T cell subsets with antibody and complement after in vitro education, completely removed the functional activity of these cell types. Addition of alpha-Ly-6.1 to MLC suppressed the MLR as well as the generation of cytotoxic lymphocytes (CTL), whereas the presence of the antibody during a cell-mediated lympholysis (CML) had no effect. Therefore, it appears that alpha-Ly-6.1 recognizes an antigen that is important for the generation of TH and TS cell subsets.