Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation.

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

Bacterial rhomboid proteases mediate quality control of orphan membrane proteins.

Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic rhomboids has remained enigmatic. Here, we show that the Shigella sonnei rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.

iRhom2 regulates ERBB signalling to promote KRAS-driven tumour growth of lung cancer cells.

Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer. Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhom proteins, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17. Here, we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 (also known as RHBDF2) to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing the cytoplasmic domain of iRhom2 as a central component of a positive feedback loop in lung cancer cells. This article has an associated First Person interview with the first authors of the paper.

Substrates and physiological functions of secretase rhomboid proteases.

Rhomboids are conserved intramembrane serine proteases with widespread functions. They were the earliest discovered members of the wider rhomboid-like superfamily of proteases and pseudoproteases. The secretase class of rhomboid proteases, distributed through the secretory pathway, are the most numerous in eukaryotes, but our knowledge of them is limited. Here we aim to summarise all that has been published on secretase rhomboids in a concise encyclopaedia of the enzymes, their substrates, and their biological roles. We also discuss emerging themes of how these important enzymes are regulated.

Extracellular cleavage of E-cadherin promotes epithelial cell extrusion.

Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main component of adherens junctions, has been shown to be essential for epithelial cell extrusion, but its mechanistic contribution remains unclear. Here, we provide clear evidence that cell extrusion can be driven by the cleavage of E-cad, both in a wild-type and an oncogenic environment. We first show that CDC42 activation in a single epithelial cell results in its efficient matrix metalloproteinase (MMP)-sensitive extrusion through MEK signalling activation and this is supported by E-cad cleavage. Second, using an engineered cleavable form of E-cad, we demonstrate that, by itself, truncation of extracellular E-cad at the plasma membrane promotes apical extrusion. We propose that extracellular cleavage of E-cad generates a rapid change in cell-cell adhesion that is sufficient to drive apical cell extrusion. Whereas in normal epithelia, extrusion is followed by apoptosis, when combined with active oncogenic signalling, it is coupled to cell proliferation.

The multiple facets of the Golgi reassembly stacking proteins.

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

Annexin A2 at the interface between F-actin and membranes enriched in phosphatidylinositol 4,5,-bisphosphate.

Vesicle rocketing has been used as a model system for understanding the dynamics of the membrane-associated F-actin cytoskeleton, but in many experimental systems is induced by persistent, non-physiological stimuli. Localised changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in membranes stimulate the recruitment of actin-remodelling proteins to their sites of action, regulate their activity and favour vesicle rocketing. The calcium and anionic phospholipid-binding protein annexin A2 is necessary for macropinocytic rocketing and has been shown to bind both PI(4,5)P2 and the barbed-ends of F-actin filaments. Here we show that annexin A2 localises to the comet tails which form constitutively in fibroblasts from patients with Lowe Syndrome. These fibroblasts are deficient in OCRL1, a phosphatidylinositol polyphosphate 5-phosphatase with specificity for PI(4,5)P2. We show that upon depletion of annexin A2 from these cells vesicle rocketing is reduced, and that this is also dependent upon PI(4,5)P2 formation. Annexin A2 co-localised with comet-tails induced by pervanadate and hyperosmotic shock in a basophilic cell line, and in an epithelial cell line upon activation of PKC. In vitro annexin A2 promoted comet formation in a bead-rocketing assay and was sufficient to link F-actin filaments to PI(4,5)P2 containing vesicles. These observations are consistent with a role for annexin A2 as an actin nucleator on PI(4,5)P2-enriched membranes.

A Simple Cell-Based Assay for the Detection of Surface Protein Shedding by Rhomboid Proteases.

Rhomboids are intramembrane serine proteases that cleave their substrates within or immediately adjacent to their transmembrane domains, a process known as regulated intramembrane proteolysis. In eukaryotes, two main types of rhomboid proteases can be distinguished based on their subcellular localization: mitochondrial rhomboids and secretase-type rhomboids that target the secretory pathway. The latter class can cleave and release the extracellular domain of all epidermal growth factor-like proteins in Drosophila and can liberate epidermal growth factor (EGF) in mammals, in a process known as ectodomain shedding. These released EGFs can then activate the EGF receptor (EGFR). EGFR signaling is crucial for mammalian development and is often deregulated in human cancer. Here we describe a cell-based protocol for detecting the ability of rhomboid proteases to release EGFR ligands into the medium. First, cells are transfected with the corresponding protease- and substrate-expressing vectors; second, cells condition the medium and accumulate shed protein. After this, protein lysates from cells and media are prepared and Western blotting is performed to detect the EGFR ligands that have been released into the medium.

FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex.

Many intercellular signals are synthesised as transmembrane precursors that are released by proteolytic cleavage ('shedding') from the cell surface. ADAM17, a membrane-tethered metalloprotease, is the primary shedding enzyme responsible for the release of the inflammatory cytokine TNFα and several EGF receptor ligands. ADAM17 exists in complex with the rhomboid-like iRhom proteins, which act as cofactors that regulate ADAM17 substrate shedding. Here we report that the poorly characterised FERM domain-containing protein FRMD8 is a new component of the iRhom2/ADAM17 sheddase complex. FRMD8 binds to the cytoplasmic N-terminus of iRhoms and is necessary to stabilise iRhoms and ADAM17 at the cell surface. In the absence of FRMD8, iRhom2 and ADAM17 are degraded via the endolysosomal pathway, resulting in the reduction of ADAM17-mediated shedding. We have confirmed the pathophysiological significance of FRMD8 in iPSC-derived human macrophages and mouse tissues, thus demonstrating its role in the regulated release of multiple cytokine and growth factor signals.

Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling.

Proteolytic cleavage and release from the cell surface of membrane-tethered ligands is an important mechanism of regulating intercellular signalling. TACE is a major shedding protease, responsible for the liberation of the inflammatory cytokine TNFα and ligands of the epidermal growth factor receptor. iRhoms, catalytically inactive members of the rhomboid-like superfamily, have been shown to control the ER-to-Golgi transport and maturation of TACE. Here, we reveal that iRhom2 remains associated with TACE throughout the secretory pathway, and is stabilised at the cell surface by this interaction. At the plasma membrane, ERK1/2-mediated phosphorylation and 14-3-3 protein binding of the cytoplasmic amino-terminus of iRhom2 alter its interaction with mature TACE, thereby licensing its proteolytic activity. We show that this molecular mechanism is responsible for triggering inflammatory responses in primary mouse macrophages. Overall, iRhom2 binds to TACE throughout its lifecycle, implying that iRhom2 is a primary regulator of stimulated cytokine and growth factor signalling.

A prospective, open-label trial of memantine in the treatment of cognitive, behavioral, and memory dysfunction in pervasive developmental disorders.

BACKGROUND: This pilot study examined the effectiveness of memantine hydrochloride in improving cognitive functioning and behavioral symptoms in children with pervasive developmental disorders (PDDs). METHOD: Subjects aged 3-12 years inclusive were enrolled in this 8-week, open-label study. Expressive and receptive language, nonverbal IQ, and nonverbal memory measures were administered at baseline and after 8 weeks of treatment with 0.4 mg/kg of memantine hydrochloride. Throughout the study, the Aberrant Behavior Checklist (ABC) was sent in weekly by parents as a measure of behavioral change. RESULTS: Twelve of 14 subjects completed the study. Significant improvement from baseline was noted on the memory test (Children's Memory Scale Dot Learning Subtest). There were no significant differences from baseline on measures of expressive or receptive language or nonverbal IQ. There were significant improvements on a number of ABC subscales, including hyperactivity, lethargy, and irritability. There were no overall significant statistical differences from baseline on the Clinical Global Improvement-Severity (CGI-S) scale. On the Clinical Global Improvement-Improvement (CGI-I), 4 of 14 subjects showed minimal improvement, and none was deemed "much-improved" or "very much improved." CONCLUSIONS: This small, prospective, open-label study suggests that memantine may be useful in the treatment of memory functioning and some behavioral symptoms in PDDs. The investigators did not see the same degree of change as endorsed by caretakers. Controlled studies are needed to substantiate and clarify these preliminary findings.

The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions.

Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue.

Genetic interaction implicates iRhom2 in the regulation of EGF receptor signalling in mice.

iRhoms are closely related to rhomboid intramembrane proteases but lack catalytic activity. In mammals iRhoms are known to regulate the trafficking of TACE, the protease that cleaves the membrane bound inflammatory cytokine TNF. We have mapped a spontaneously occurring mouse mutation with a loss of hair phenotype, curly bare (cub), to the Rhbdf2 locus, which encodes the iRhom2 protein. The cub deletion removes the first 268 amino acids of the iRhom2 protein but is not a loss of function. We have also identified a previously reported suppressor of cub, called Mcub (modifier of curly bare), and find it to be a loss of function allele of the amphiregulin gene (Areg). Amphiregulin is an activating ligand of the epidermal growth factor receptor (EGFR) that, like TNF, is released by TACE. Our results therefore imply a regulatory link between iRhoms and EGFR signalling in mammals. We have tested the model that the cub mutation leads to iRhom2 hyperactivity and consequently excess TACE processing of amphiregulin and elevated EGFR signalling. Our results do not support this hypothesis: we find that, compared to wild-type cells, cub mutant embryonic fibroblasts release less amphiregulin, and that the cub mutant form of iRhom2 is less able than wild type to bind to TACE and promote its maturation.

GRASP65 controls the cis Golgi integrity in vivo.

GRASP65 and GRASP55 are peripheral Golgi proteins localized to cis and medial/trans cisternae, respectively. They are implicated in diverse aspects of protein transport and structure related to the Golgi complex, including the stacking of the Golgi stack and/or the linking of mammalian Golgi stacks into the Golgi ribbon. Using a mouse model, we interfered with GRASP65 by homologous recombination and confirmed its absence of expression. Surprisingly, the mice were healthy and fertile with no apparent defects in tissue, cellular or subcellular organization. Immortalized MEFs derived from the mice did not show any growth or morphological defects. However, despite the normal appearance of the Golgi ribbon, a fluorescence recovery after photobleaching assay revealed functional discontinuities specific to the cis cisternal membrane network. This leads to a strong change in the plasma membrane GSII lectin staining that was also observed in certain mutant tissues. These findings substantiate the role of GRASP65 in continuity of the cis Golgi network required for proper glycosylation, while showing that neither this continuity nor GRASP65 itself are essential for the viability of a complex organism.

Long-term follow-up of cystic fibrosis newborn screening: psychosocial functioning of adolescents and young adults.

BACKGROUND: Long-term psychosocial outcomes of cystic fibrosis (CF) patients diagnosed through newborn screening remain unknown. METHODS: This cross-sectional study compared three groups of youths (16 to 22 years): CF patients diagnosed through NBS (CF-NBS, n = 13), CF patients diagnosed through standard practice (CF-SP, n = 26) and healthy peers (H, n = 42), plus 72 of their parents. We hypothesized that adolescent psychological functioning would be mediated by parent depression and quality of parent-child communication and cohesiveness. RESULTS: A path analysis showed significantly more depression among CF-NBS group parents (p = .006-.008). Parent-child cohesiveness was related to communication (p < .001). Cohesiveness and communication were associated with youth Internalizing Problems (p = .037, p = .009), Emotional Symptoms (p = 0.018, p = 0.022), and Personal Adjustment (communication only, p = 0.009). Parent depression was related to youth Personal Adjustment (p = 0.022). CONCLUSIONS: CF patients report psychosocial function similar to healthy peers. Parents of children diagnosed with CF through NBS may be at risk for depressive symptoms when their children reach adolescence.

Neurocognitive function and state cognitive stress appraisal predict cortisol reactivity to an acute psychosocial stressor in adolescents.

Stress and associated alterations in hypothalamic-pituitary-adrenal (HPA) function have deleterious influence on the development of multiple mental and physical health problems. Prior research has aimed to identify individuals most at risk for the development of these stress-related maladies by examining factors that may contribute to inter-individual differences in HPA responses to acute stress. The objectives of this study were to investigate, in adolescents, (1) whether differences in neurocognitive abilities influenced cortisol reactivity to an acute stressor, (2) whether internalizing psychiatric disorders influenced this relationship, and (3) whether acute cognitive stress-appraisal mechanisms mediated an association between neurocognitive function and cortisol reactivity. Subjects were 70 adolescents from a community sample who underwent standardized neurocognitive assessments of IQ, achievement, and declarative memory measures at mean age 14 and whose physiological and behavioral responses to a standardized psychosocial stress paradigm (Trier Social Stress Test, TSST) were assessed at mean age 18. Results showed that, among all adolescents, lower nonverbal memory performance predicted lower cortisol reactivity. In addition, internalizing disorders interacted with verbal memory such that the association with cortisol reactivity was strongest for adolescents with internalizing disorders. Finally, lower secondary cognitive appraisal of coping in anticipation of the TSST independently predicted lower cortisol reactivity but did not mediate the neurocognitive-cortisol relationship. Findings suggest that declarative memory may contribute to inter-individual differences in acute cortisol reactivity in adolescents, internalizing disorders may influence this relationship, and cognitive stress appraisal also predicts cortisol reactivity. Developmental, research, and clinical implications are discussed.

Health-related quality of life in children and adolescents with cystic fibrosis: convergent validity with parent-reports and objective measures of pulmonary health.

OBJECTIVE: This study examined the convergent validity of health-related quality of life (HRQOL) reported by patients with cystic fibrosis compared with their parents' reports and objective pulmonary measures across 3 time points. METHODS: Ninety-two children (8-13 years) and adolescents (14-18 years) with cystic fibrosis and their parents completed Cystic Fibrosis Questionnaires to examine concordance with Wisconsin chest x-ray (WCXR) scores and pulmonary function tests, for example, forced expiratory volume at 1 second (FEV1), and parent-child/adolescent concordance across multiple HRQOL domains. Concordance was analyzed relative to patient age and gender. RESULTS: Parent-reports were closely aligned with WCXR scores, whereas patient reports were more closely aligned with FEV1. Adolescents and parents of both age groups had more HRQOL domains concordant with pulmonary health measures than did child self-reports. Parent-child concordance was inversely related to child age, particularly with male adolescents. Children generally reported better HRQOL than parents. Male adolescents and their parents were more likely to have significantly discordant HRQOL scores than female adolescents and their parents. Male and female adolescents reported higher HRQOL than their parents reported for all but vitality and health perception domains. Younger male children showed concordance with their parents on 5 of 7 domains. CONCLUSIONS: Parent-child/adolescent discordance on HRQOL was consistent with normative child development expectations. Findings underscore the value of enlisting perspectives from parents as well as children regarding HRQOL.

Annexin A2 at the interface of actin and membrane dynamics: a focus on its roles in endocytosis and cell polarization.

Annexins are a family of calcium- and phospholipid-binding proteins found in nearly all eukaryotes. They are structurally highly conserved and have been implicated in a wide range of cellular activities. In this paper, we focus on Annexin A2 (AnxA2). Altered expression of this protein has been identified in a wide variety of cancers, has also been found on the HIV particle, and has been implicated in the maturation of the virus. Recently, it has also been shown to have an important role in the establishment of normal apical polarity in epithelial cells. We synthesize here the known biochemical properties of this protein and the extensive literature concerning its involvement in the endocytic pathway. We stress the importance of AnxA2 as a platform for actin remodeling in the vicinity of dynamic cellular membranes, in the hope that this may shed light on the normal functions of the protein and its contribution to disease.

Golgi bypass: skirting around the heart of classical secretion.

Classical secretion consists of the delivery of transmembrane and soluble proteins to the plasma membrane and the extracellular medium, respectively, and is mediated by the organelles of the secretory pathway, the Endoplasmic Reticulum (ER), the ER exit sites, and the Golgi, as described by the Nobel Prize winner George Palade (Palade 1975). At the center of this transport route, the Golgi stack has a major role in modifying, processing, sorting, and dispatching newly synthesized proteins to their final destinations. More recently, however, it has become clear that an increasing number of transmembrane proteins reach the plasma membrane unconventionally, either by exiting the ER in non-COPII vesicles or by bypassing the Golgi. Here, we discuss the evidence for Golgi bypass and the possible physiological benefits of it. Intriguingly, at least during Drosophila development, Golgi bypass seems to be mediated by a Golgi protein, dGRASP, which is found ectopically localized to the plasma membrane.

Unconventional secretion: a stress on GRASP.

Most proteins follow the classical secretory pathway from the endoplasmic reticulum, via the Golgi, to the plasma membrane or extracellular medium. However, some proteins reach these final destinations by two alternative routes. One sustains the extracellular delivery of cytoplasmic proteins that lack a signal peptide, the other supports the transport of transmembrane proteins to the plasma membrane in a manner that bypasses the Golgi. Here, we highlight the observation that some unconventional secretion events are triggered by cellular stress. Furthermore, one Golgi protein, Golgi Re-Assembly and Stacking Protein (GRASP), has been shown to be essential to both types of unconventional secretion and we discuss ways in which it may support these events in a Golgi-independent manner.