Laura F. Dagley

Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation

A mutation in pyrin that disrupts regulation leads to autoinflammatory disease. Guarding inflammation The innate immune system is hard-wired to protect people from infection. However, mutations in these protective genes can lead to uncontrolled inflammation, resulting in autoinflammatory disease. Now, Masters et al. describe a family with an autoinflammatory disease caused by a previously unreported mutation in pyrin. This mutation disrupts pyrin regulation and mimics the effect of pathogen sensing by pyrin, leading to proinflammatory interleukin-1β (IL-1β) production. Indeed, targeting IL-1β resolved disease in one patient. These data suggest that pyrin is regulated through a guard-like mechanism, which guards against autoinflammation in humans. Pyrin responds to pathogen signals and loss of cellular homeostasis by forming an inflammasome complex that drives the cleavage and secretion of interleukin-1β (IL-1β). Mutations in the B30.2/SPRY domain cause pathogen-independent activation of pyrin and are responsible for the autoinflammatory disease familial Mediterranean fever (FMF). We studied a family with a dominantly inherited autoinflammatory disease, distinct from FMF, characterized by childhood-onset recurrent episodes of neutrophilic dermatosis, fever, elevated acute-phase reactants, arthralgia, and myalgia/myositis. The disease was caused by a mutation in MEFV, the gene encoding pyrin (S242R). The mutation results in the loss of a 14-3-3 binding motif at phosphorylated S242, which was not perturbed by FMF mutations in the B30.2/SPRY domain. However, loss of both S242 phosphorylation and 14-3-3 binding was observed for bacterial effectors that activate the pyrin inflammasome, such as Clostridium difficile toxin B (TcdB). The S242R mutation thus recapitulated the effect of pathogen sensing, triggering inflammasome activation and IL-1β production. Successful therapy targeting IL-1β has been initiated in one patient, resolving pyrin-associated autoinflammation with neutrophilic dermatosis. This disease provides evidence that a guard-like mechanism of pyrin regulation, originally identified for Nod-like receptors in plant innate immunity, also exists in humans.

Membrane proteomics by high performance liquid chromatography–tandem mass spectrometry: Analytical approaches and challenges

Membrane proteins (MPs) play diverse biologically important structural and functional roles including molecular transport, cell communication, and signal transduction. The dysfunctions of many are linked to deleterious human diseases and thus are of utmost importance in drug discovery. MPs comprise approximately 20–30% of all open reading frames (ORFs), however they are typically under‐represented in many LC‐MS proteomics experiments due to their low abundance and poor solubility. To address these analytical challenges, various MP enrichment, solubilization, digestion, and fractionation strategies have been employed to further improve the coverage of the membrane systems while maintaining compatibility with MS detection. This review discusses both established and emerging high‐throughput gel‐free analytical workflows in membrane proteomics, and the inherent advantages, disadvantages, and orthogonality of the various approaches. The issues of critical importance for successful LC‐MS/MS detection such as detergent selection and minimizing ion suppression in detergent‐based workflows are discussed in detail. Recent studies comparing the performance of different analytical strategies are highlighted in order to provide practical insight into the choice of the most appropriate method for membrane‐centric applications ranging from cell surface biomarker discovery to MP interaction network mapping.

CIS is a potent checkpoint in NK cell–mediated tumor immunity

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish−/− mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell–mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling1–3. RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis4,5. Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

Regulation of Starch Stores by a Ca2+-Dependent Protein Kinase Is Essential for Viable Cyst Development in Toxoplasma gondii

Transmissible stages of Toxoplasma gondii store energy in the form of the carbohydrate amylopectin. Here, we show that the Ca(2+)-dependent protein kinase CDPK2 is a critical regulator of amylopectin metabolism. Increased synthesis and loss of degradation of amylopectin in CDPK2 deficient parasites results in the hyperaccumulation of this sugar polymer. A carbohydrate-binding module 20 (CBM20) targets CDPK2 to amylopectin stores, while the EF-hands regulate CDPK2 kinase activity in response to Ca(2+) to modulate amylopectin levels. We identify enzymes involved in amylopectin turnover whose phosphorylation is dependent on CDPK2 activity. Strikingly, accumulation of massive amylopectin granules in CDPK2-deficient bradyzoite stages leads to gross morphological defects and complete ablation of cyst formation in a mouse model. Together these data show that Ca(2+) signaling regulates carbohydrate metabolism in Toxoplasma and that the post-translational control of this pathway is required for normal cyst development.

Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients

Objectives We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). Methods Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. Results We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. Conclusions A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS.

Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling

Influenza virus infections have a significant impact on global human health. Individuals with suppressed immunity, or suffering from chronic inflammatory conditions such as COPD, are particularly susceptible to influenza. Here we show that suppressor of cytokine signaling (SOCS) five has a pivotal role in restricting influenza A virus in the airway epithelium, through the regulation of epidermal growth factor receptor (EGFR). Socs5-deficient mice exhibit heightened disease severity, with increased viral titres and weight loss. Socs5 levels were differentially regulated in response to distinct influenza viruses (H1N1, H3N2, H5N1 and H11N9) and were reduced in primary epithelial cells from COPD patients, again correlating with increased susceptibility to influenza. Importantly, restoration of SOCS5 levels restricted influenza virus infection, suggesting that manipulating SOCS5 expression and/or SOCS5 targets might be a novel therapeutic approach to influenza. DOI: http://dx.doi.org/10.7554/eLife.20444.001

Application of quantitative proteomics technologies to the biomarker discovery pipeline for multiple sclerosis.

Multiple sclerosis is an inflammatory‐mediated demyelinating disorder most prevalent in young Caucasian adults. The various clinical manifestations of the disease present several challenges in the clinic in terms of diagnosis, monitoring disease progression and response to treatment. Advances in MS‐based proteomic technologies have revolutionized the field of biomarker research and paved the way for the identification and validation of disease‐specific markers. This review focuses on the novel candidates discovered by the application of quantitative proteomics to relevant disease‐affected tissues in both the human context and within the animal model of the disease known as experimental autoimmune encephalomyelitis. The role of targeted MS approaches for biomarker validation studies, such as multiple reaction monitoring will also be discussed.

Quantitative proteomic profiling reveals novel region‐specific markers in the adult mouse brain

Despite major advances in neuroscience, a comprehensive understanding of the structural and functional components of the adult brain compartments remains to be fully elucidated at a quantitative molecular level. Indeed, over half of the soluble‐ and membrane‐annotated proteins are currently unmapped within online digital brain atlases. In this study, two complementary approaches were used to assess the unique repertoire of proteins enriched within select regions of the adult mouse CNS, including the brain stem, cerebellum, and remaining brain hemispheres. Of the 1200 proteins visualized by 2D‐DIGE, approximately 150 (including cytosolic and membrane proteins) were found to exhibit statistically significant changes in relative abundance thus representing putative region‐specific brain markers. In addition to using a high‐precision 18O‐labeling strategy for the quantitative LC‐MS/MS mapping of membrane proteins isolated from myelin‐enriched fractions, we have identified over 1000 proteins that have yet to be described in any other mammalian myelin proteome. A comparison of our myelin proteome was made to an existing transcriptome database containing mRNA abundance profiles during oligodendrocyte differentiation and has confirmed statistically significant abundance changes for ∼500 of these newly mapped proteins, thus revealing new roles in oligodendrocyte and myelin biology. These data offer a resource for the neuroscience community studying the molecular basis for specialized neuronal activities in the CNS and myelin‐related disorders. The MS proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000327 (http://proteomecentral.proteomexchange.org/dataset/PXD000327).

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N-Terminus of SOCS5.

SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements, and NOE analysis, we demonstrate the presence of preformed structural elements in the JIR of mouse SOCS5 (mSOCS5175-244), consisting of an α-helix encompassing residues 224-233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis.