BEACH Domain Research Articles

LRBA, a BEACH protein mutated in human immune deficiency, is widely expressed in epithelia, exocrine and endocrine glands, and neurons

Mutations in LRBA, a BEACH domain protein, cause severe immune deficiency in humans. LRBA is expressed in many tissues and organs according to biochemical analysis, but little is known about its cellular and subcellular localization, and its deficiency phenotype outside the immune system. By LacZ histochemistry of Lrba gene-trap mice, we performed a comprehensive survey of LRBA expression in numerous tissues, detecting it in many if not all epithelia, in exocrine and endocrine cells, and in subpopulations of neurons. Immunofluorescence microscopy of the exocrine and endocrine pancreas, salivary glands, and intestinal segments, confirmed these patterns of cellular expression and provided information on the subcellular localizations of the LRBA protein. Immuno-electron microscopy demonstrated that in neurons and endocrine cells, which co-express LRBA and its closest relative, neurobeachin, both proteins display partial association with endomembranes in complementary, rather than overlapping, subcellular distributions. Prominent manifestations of human LRBA deficiency, such as inflammatory bowel disease or endocrinopathies, are believed to be primarily due to immune dysregulation. However, as essentially all affected tissues also express LRBA, it is possible that LRBA deficiency enhances their vulnerability and contributes to the pathogenesis.

NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells

Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease.

Identification of novel variants in a Chinese patient with Chediak-Higashi syndrome

To explore the genetic basis for a child featuring Chediak-Higashi syndrome (CHS). Clinical manifestations and results of auxiliary examination of the proband were analyzed. The proband was subjected to whole exome sequencing, and the results were verified by Sanger sequencing. Correlation between the genotype and clinical phenotype was analyzed. The proband showed partial skin albinism, recurrent respiratory infection and other immune deficiencies. Genetic testing showed that he has harbored c.2437C>T (p.Arg813*) and c.6077dupA (p.Tyr2026fs) (NM_000081) compound heterozygous variants of the LYST gene, for which his parents were both carriers. Neither variant was reported previously. HEAT repeats domain was frequently associated with more severe phenotype of CHS (81.6%), whilst no variant has been found in the PH_BEACH domain. This study has enriched the spectrum of LYST gene variants associated with CHS and enabled clinical diagnosis, prenatal diagnosis and prognostic evaluation for the child.

Wave Enhanced Overtides in an Idealized Tidal Inlet System

AbstractTidal water levels and currents in the shallow water environments of the inner shelf are subject to nonlinear interactions that produce higher frequency harmonics known as overtides, which contribute to the residual transport of scalars. The primary hydrodynamic overtide generating mechanisms (advection, nonlinear continuity and bottom friction) are well known. Yet, other mechanisms may exist in coupled systems characterized by wind waves and barotropic tides. This work uses process‐oriented coupled tides‐waves numerical simulations of an idealized barrier beach domain to study the effect of varying tidal range and wave conditions (height, period, direction) to investigate new mechanisms of overtide generation. Harmonic analysis of results from 92 simulations indicates that an increase in wave height amplifies overtide current velocities in both the alongshore and cross‐shore directions, while changes in wave period and wave direction have a lesser effect. A maximum increase of ∼140% in overtide current velocity magnitudes was observed in domain‐wide averages from a tides only to a coupled tides‐waves simulation. Decomposition of the depth‐averaged momentum balance of the tides only and tides‐waves simulations identify bottom stress, pressure gradient, and bottom wave streaming enhancing overtide generation. Accelerations from wave‐enhanced pressure gradients and bottom wave streaming produce a shoreward bottom current and an offshore directed return flow that persist throughout a semidiurnal tidal cycle, interacting with tidal currents to enhance asymmetries between flood and ebb.

Clinically Complex LRBA Deficiency Due to a Founder Allele in the Georgian Jewish Population.

Pathogenic variants in LRBA, encoding the LPS Responsive Beige-Like Anchor (LRBA) protein, are responsible for recessive, early-onset hypogammaglobulinemia, severe multi-organ autoimmunity, and lymphoproliferation, with increased risk for malignancy. LRBA deficiency has a wide clinical spectrum with variable age of onset and disease severity. Three apparently unrelated patients with LRBA deficiency, of Georgian Jewish descent, were homozygous for LRBA c.6640C > T, p.R2214*, leading to a stop upstream of the LRBA BEACH domain. Despite carrying the same LRBA genotype, the three patients differed in clinical course: the first patient was asymptomatic until age 25years; the second presented with failure to thrive at age 3months; and the third presented at age 7years with immune cytopenias and severe infections. Two of the patients developed malignancies: the first patient was diagnosed with recurrent Hodgkin's disease at age 36years, and the second patient developed aggressive gastric cancer at age 15years. Among Georgian Jews, the carrier frequency of the LRBA p.R2214* allele was 1.6% (4 of 236 Georgian Jewish controls). The allele was absent from other populations. Haplotype analysis showed a shared origin of the mutation. These three patients revealed a pathogenic LRBA founder allele in the Georgian Jewish population, support the diverse and complex clinical spectrum of LRBA deficiency, and support the possibility that LRBA deficiency predisposes to malignancy.

A null allele in the wdfy-3 selective autophagy gene of C. elegans .

The C. elegans WDFY-3 protein is important for cargo selection during selective autophagy and for regulating axon termination. The C-terminal region of WDFY-3 contains BEACH, WD repeats, and FYVE-like domains, all of which are required for selective autophagy. WDFY-3 also contains a large N-terminal region that is relatively uncharacterized. Currently, wdfy-3(ok912) is the only mutant allele that has been characterized for this gene. This allele features a small deletion that is predicted to disrupt the C-terminal region of the protein. Here, we used CRISPR Cas9 to produce a new wdfy-3(cue30) allele that is a near complete deletion of the coding region. We report that, unlike the existing wdfy-3(ok912) allele, this new wdfy-3(cue30) null allele causes a weak overextension phenotype in the PLM axon. Like the existing wdfy-3(ok912) allele, the new wdfy-3(cue30) null allele can suppress PLM axon termination defects caused by an fsn-1 null allele. Creating and characterizing new wdfy-3 alleles will increase our understanding of this gene and could help elucidate more of the gene’s conserved functions.

Reduction of WDR81 impairs autophagic clearance of aggregated proteins and cell viability in neurodegenerative phenotypes.

Neurodegenerative diseases are characterized by neuron loss and accumulation of undegraded protein aggregates. These phenotypes are partially due to defective protein degradation in neuronal cells. Autophagic clearance of aggregated proteins is critical to protein quality control, but the underlying mechanisms are still poorly understood. Here we report the essential role of WDR81 in autophagic clearance of protein aggregates in models of Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD). In hippocampus and cortex of patients with HD, PD and AD, protein level of endogenous WDR81 is decreased but autophagic receptor p62 accumulates significantly. WDR81 facilitates the recruitment of autophagic proteins onto Htt polyQ aggregates and promotes autophagic clearance of Htt polyQ subsequently. The BEACH and MFS domains of WDR81 are sufficient for its recruitment onto Htt polyQ aggregates, and its WD40 repeats are essential for WDR81 interaction with covalent bound ATG5-ATG12. Reduction of WDR81 impairs the viability of mouse primary neurons, while overexpression of WDR81 restores the viability of fibroblasts from HD patients. Notably, in Caenorhabditis elegans, deletion of the WDR81 homolog (SORF-2) causes accumulation of p62 bodies and exacerbates neuron loss induced by overexpressed α-synuclein. As expected, overexpression of SORF-2 or human WDR81 restores neuron viability in worms. These results demonstrate that WDR81 has crucial evolutionarily conserved roles in autophagic clearance of protein aggregates and maintenance of cell viability under pathological conditions, and its reduction provides mechanistic insights into the pathogenesis of HD, PD, AD and brain disorders related to WDR81 mutations.

NBEAL1 controls SREBP2 processing and cholesterol metabolism and is a susceptibility locus for coronary artery disease

Dysregulated cholesterol homeostasis promotes the pathology of atherosclerosis, myocardial infarction and strokes. Cellular cholesterol is mainly regulated at the transcriptional level by SREBP2, but also through uptake of extracellular cholesterol from low density lipoproteins (LDL) via expression of LDL receptors (LDLR) at the cell surface. Identification of the mechanisms involved in regulation of these processes are thus key to understand the pathology of coronary artery disease. Here, we identify the large and poorly characterized BEACH domain protein Neurobeachin-like (NBEAL) 1 as a Golgi- associated protein required for regulation of cholesterol metabolism. NBEAL1 is most abundantly expressed in arteries. Genetic variants in NBEAL1 are associated with decreased expression of NBEAL1 in arteries and increased risk of coronary artery disease in humans. We show that NBEAL1 regulates cholesterol metabolism by modulating LDLR expression in a mechanism involving interaction with SCAP and PAQR3 and subsequent SREBP2-processing. Thus, low expression of NBEAL1 may lead to increased risk of coronary artery disease by downregulation of LDLR levels.

A Spectrum of Clinical Findings from ALPS to CVID: Several Novel LRBA Defects.

Autosomal recessively inherited lipopolysaccharide-responsive beige-like anchor (LRBA) protein deficiency was shown to be responsible for different types of inborn errors of immunity, such as common variable immunodeficiency (CVID) and autoimmune lymphoproliferative syndrome (ALPS). The aim of this study was to compare patients with LRBA-related ALPS and LRBA-related CVID, to describe their clinical and laboratory phenotypes, and to prepare an algorithm for their diagnosis and management. Fifteen LRBA-deficient patients were identified among 31 CVID and 14 possible ALPS patients with Western blotting (WB), primary immunodeficiency disease (PIDD) gene, next-generation panel screening (NGS), and whole exome sequencing (WES). The median age on admission and age of diagnosis were 7years (0.3-16.5) and 11years (5-44), respectively. Splenomegaly was seen in 93.3% (14/15) of the patients on admission. Splenectomy was performed to 1/5. Recurrent upper respiratory tract infections (93.3% (14/15)), autoimmune cytopenia (80% (12/15)), chronic diarrhea (53.3% (8/15)), lower respiratory tract infections (53.3% (8/15)), lymphoma (26.6% (4/15)), Evans syndrome (26.6% (4/15)), and autoimmune thyroiditis (20% (3/15)) were common clinical findings and diseases. Lymphopenia (5/15), intermittant neutropenia (4/15), eosinophilia (4/15), and progressive hypogammaglobulinemia are recorded in given number of patients. Double negative T cells (TCRαβ+CD4-CD8-) were increased in 80% (8/10) of the patients. B cell percentage/numbers were low in 60% (9/15) of the patients on admission. Decreased switched memory B cells, decreased naive and recent thymic emigrant (RTE) Thelper (Th) cells, markedly increased effector memory/effector memory RA+ (TEMRA) Th were documented. Large PD1+ population, increased memory, and enlarged follicular helper T cell population in the CD4+ T cell compartment was seen in one of the patients. Most of the deleterious missense mutations were located in the DUF1088 and BEACH domains. Interestingly, one of the two siblings with the same homozygous LRBA defect did not have any clinical symptom. Hematopoietic stem cell transplantation (HSCT) was performed to 7/15 (46.6%) of the patients. Transplanted patients are alive and well after a median of 2years (1-3). In total, one patient died from sepsis during adulthood before HSCT. Patients with LRBA deficiency may initially be diagnosed as CVID or ALPS in the clinical practice. Progressive decrease in B cells as well as IgG in ALPS-like patients and addition of IBD symptoms in the follow-up should raise the suspicion for LRBA deficiency. Decreased switched memory B cells, decreased naive and recent thymic emigrant (RTE) Th cells, and markedly increased effector memory/effector memory RA+ Th cells (TEMRA Th) cells are important for the diagnosis of the patients in addition to clinical features. Analysis of protein by either WB or flow cytometry is required when the clinicians come across especially with missense LRBA variants of uncertain significance. High rate of malignancy shows the regulatory T cell's important role of immune surveillance. HSCT is curative and succesful in patients with HLA-matched family donor.

The BEACH Domain Is Critical for Blue Cheese Function in a Spatial and Epistatic Autophagy Hierarchy.

Drosophila blue cheese (bchs) encodes a BEACH domain adaptor protein that, like its human homolog ALFY, promotes clearance of aggregated proteins through its interaction with Atg5 and p62. bchs mutations lead to age-dependent accumulation of ubiquitinated inclusions and progressive neurodegeneration in the fly brain, but neither the influence of autophagy on bchs-related degeneration, nor bchs’ placement in the autophagic hierarchy have been shown. We present epistatic evidence in a well-defined larval motor neuron paradigm that in bchs mutants, synaptic accumulation of ubiquitinated aggregates and neuronal death can be rescued by pharmacologically amplifying autophagic initiation. Further, pharmacological rescue requires at least one intact BEACH-containing isoform of the two identified in this study. Genetically augmenting a late step in autophagy, however, rescues even a strong mutation which retains only a third, non-BEACH containing isoform. Using living primary larval brain neurons, we elucidate the primary defect in bchs to be an excess of early autophagic compartments and a deficit in mature compartments. Conversely, rescuing the mutants by full-length Bchs over-expression induces mature compartment proliferation and rescues neuronal death. Surprisingly, only the longest Bchs isoform colocalizes well with autophagosomes, and shuttles between different vesicular locations depending on the type of autophagic impetus applied. Our results are consistent with Bchs promoting autophagic maturation, and the BEACH domain being required for this function.HIGHLIGHTSThe autophagic adaptor blue cheese is placed in an epistatic hierarchy, using pharmacological and genetic modulation of bchs- motor neuron degeneration. An intact BEACH isoform can promote autophagic proliferation, and in primary larval brain neurons Bchs shuttles to different components of the autophagy machinery, dependent on the stimulus.

Nbeal2 interacts with Dock7, Sec16a, and Vac14

AbstractMutations in NBEAL2, the gene encoding the scaffolding protein Nbeal2, are causal of gray platelet syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a, and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction with Dock7 and Vac14, respectively. Proximity ligation assays show that these 2 proteins are physically proximal to Nbeal2 in human megakaryocytes. In addition, we demonstrate that Nbeal2 is primarily localized in the cytoplasm and Dock7 on the membrane of or in α-granules. Interestingly, platelets from GPS cases and Nbeal2−/− mice are almost devoid of Dock7, resulting in a profound dysregulation of its signaling pathway, leading to defective actin polymerization, platelet activation, and shape change. This study shows for the first time proteins interacting with Nbeal2 and points to the dysregulation of the canonical signaling pathway of Dock7 as a possible cause of the aberrant formation of platelets in GPS cases and Nbeal2-deficient mice.

The BEACH Protein LRBA Promotes the Localization of the Heterotrimeric G-protein Golf to Olfactory Cilia

BEACH domain proteins are involved in membrane protein traffic and human diseases, but their molecular mechanisms are not understood. The BEACH protein LRBA has been implicated in immune response and cell proliferation, and human LRBA mutations cause severe immune deficiency. Here, we report a first functional and molecular phenotype outside the immune system of LRBA-knockout mice: compromised olfaction, manifesting in reduced electro-olfactogram response amplitude, impaired food-finding efficiency, and smaller olfactory bulbs. LRBA is prominently expressed in olfactory and vomeronasal chemosensory neurons of wild-type mice. Olfactory impairment in the LRBA-KO is explained by markedly reduced concentrations (20–40% of wild-type levels) of all three subunits αolf, β1 and γ13 of the olfactory heterotrimeric G-protein, Golf, in the sensory cilia of olfactory neurons. In contrast, cilia morphology and the concentrations of many other proteins of olfactory cilia are not or only slightly affected. LRBA is also highly expressed in photoreceptor cells, another cell type with a specialized sensory cilium and heterotrimeric G-protein-based signalling; however, visual function appeared unimpaired by the LRBA-KO. To our knowledge, this is the first observation that a BEACH protein is required for the efficient subcellular localization of a lipid-anchored protein, and of a ciliary protein.

Polymorphonuclears Display a New Type of Abnormal Cytologic Granules (Chediak Higashi-Like) in a Very Rare Syndrome Linked to a Biallelic Defect of WDR81

A twelve years-old girl was admitted at the pediatric emergency unit for a severe pleuro-pneumopathy. She had a long history of recurrent infections in a complex neurological context. Since birth, she had suffered from an epileptic encephalopathy with West syndrome, severe microcephaly and spastic tetraplegia. Her neurological development was extremely impaired: she was not able to neither hold her head nor develop any voluntary hand use. Moreover, she had a precocious puberty and a progressive worsening of her neurological status. She is bedridden, has very poor visual contact and does not speak.Brain magnetic resonance imaging (MRI) examination has evidenced a severe microcephaly, without gyral anomalies, cerebral atrophy predominating in the frontal lobes, hypoplasia of the corpus callosum and dysmyelination.Her older sister and parents are healthy and there is no history of consanguinity in the family.During the infection mentioned above, the only anomaly at complete blood count (CBC) was an excess of monocytes. Yet, and although the CBC instrument had not indicated any alarm for neutrophils, abnormal granules were observed microscopically in polymorphonuclears on the blood smear (fig. 1). The coarse and purplish granules were toluidine-negative, myeloperoxidase-positive and only present in the polymorphonuclear lineage. The large vacuoles in these cells, also seen in monocytes, are related to the infectious condition of the patient in this context of pleuro-pneumopathy. There was no anomaly of the lymphocytes, and specifically no image recalling storage disease.Although previous CBC had not led to the identification of these granules, they were systematically investigated for, at high magnification, afterwards, always observed but seemed to increase during each infectious episode. Their appearance suggests a coalescence of smaller granules during infection.A bone marrow aspiration was performed as the patient underwent surgery for severe scoliosis, at distance from any infectious episode. Abnormal granules were present at all stages of neutrophil maturation (fig. 2).Nobody in the family presents these abnormal granules. Yet, investigations were performed in search of a congenital syndrome or storage disease: metabolic balance, amino acids chromatography in blood and urine, functional analyses of polymorphonuclears, karyotype, CGH array. No anomaly was disclosed. A skin biopsy looking for inclusions allowed to exclude lipofuscinose. Several other investigations were performed which excluded a lysosomal storage disease.A whole exome analysis was then decided for the child, parents and sister after obtaining informed consent from the parents. This allowed to discover mutations on both alleles ofthe WDR81 gene in the propositus: a deletion leading to a frame shift in exon 3 and a substitution generating a missense in exon 9 (c.3820_3835del p.Pro1274Thrfs*56 and c.5453G>T p.Gly1818Val respectively). The c.3820_3835del deletion was inherited by the father and the c.5453G>T mutation was inherited by the mother.The mutations were confirmed by Sanger sequencing and segregated with the expected pattern of autosomal-recessive inheritance in all available family members.This gene has previously been shown to be associated with cerebral ataxia, intellectual disability and quadrupedal locomotion in patients with homozygous mutations in consanguineous families. Moreover, a murine model with mutation of this gene showed tremor and ataxic gait. Expression of WDR81 was found in neuron of central nervous system included Purkinje cells, photoreceptor cells. All these aspects are consistent with the clinical features of the patient, the severity of her disease being possibly related to the fact that both alleles of the WDR81 gene carry a different mutation.This also suggests that the double genetic defect observed in the WDR81 in this child is responsible for the peculiar granules appearing early during myeloid maturation. Interestingly, the WDR81 gene contains a BEACH domain on its N terminal portion. This domain was described in the BEIGE protein and the highly homologous CHS protein which are involved in Chediak-Higashi syndromes. Although the patient has none of the phenotypic nor immune characteristics of a Chediak-Higashi syndrome, it is highly likely that involvement of the WDR81 gene is responsible for the formation of such pseudo Chediak abnormal granules. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription.

BackgroundNeurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed.MethodsYeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression.ResultsBoth domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated.ConclusionDefining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for neural development.

A Caenorhabditis elegans Homologue of LYST Functions in Endosome and Lysosome-Related Organelle Biogenesis.

LYST-1 is a Caenorhabditis elegans BEACH domain containing protein (BDCP) homologous to LYST and NBEAL2, BDCPs controlling organelle biogenesis that are implicated in human disease. Unlike the three other BDCPs encoded by C. elegans, mutations in lyst-1 lead to smaller lysosome-related organelles (LROs), smaller lysosomes, increased numbers of LROs and decreased numbers of early endosomes. lyst-1(-) mutations do not obviously disrupt protein trafficking to lysosomes or LROs, however, the formation of gut granules is diminished.

Far from Membranes, BEACH Domains Regulate Stress-Related mRNAs.

Sometimes in research, an unexpected finding can not only lead to an advance in one field but also shed light on a mystery in another. That appears to be the case in a new study from Alexandra Steffens, Martin Huelskamp, and colleagues, who show that a protein previously believed to be exclusively involved in membrane dynamics also plays a critical role in regulating salt stress responses in plants. Because this stress response function depends on a domain that is widely distributed and highly conserved throughout eukaryotic life, it seems likely that this domain plays a similar stress-response role elsewhere, explaining previously mysterious consequences of mutations in related proteins in mice. The protein in question, from the model plant Arabidopsis, is called SPIRRIG and contains the highly conserved BEACH domain. Proteins containing these domains have been known as regulators of various membrane trafficking events, and the authors indeed found a number of membrane-specific binding partners when they performed a binding assay using SPIRRIG's BEACH domain as bait. But they also discovered the domain bound to a nonmembrane protein called decapping protein 1 (DCP1), a core component of P-bodies. They confirmed the result with the entire SPIRRIG protein and showed it was specific for DCP1, not any of several other P-body proteins. P-bodies are highly dynamic cytoplasmic structures that sequester messenger RNA, thereby temporarily limiting protein synthesis, a common response to various types of stress. To test whether SPIRRIG was involved in stress responses in Arabidopsis, the authors subjected leaves to osmotic stress with either salt or the sugar mannitol. Mannitol had no effect, but salt triggered accumulation of SPIRRIG in P-bodies. Mutations of SPIRRIG resulted in a reduced P-body formation, and mutant plants grew poorly in the presence of salt compared to their wild-type relatives (Fig 1). Fig 1 Loss of the SPIRRIG protein leads to salt stress hypersensitivity in Arabidopsis thaliana. At the subcellular level, SPIRRIG mutation in the presence of salt stress affected the expression of multiple genes and the stability of multiple RNA transcripts, many of which, but not all, were directly involved in salt tolerance. Mutation of SPIRRIG reduced sequestration of its mRNA targets into RNA granules and ultimately into P-bodies. The discovery of a salt stress regulator in Arabidopsis has potential significance in understanding how plants, including agriculturally important ones, are likely to adapt to a world in which changing rainfall patterns will likely increase soil salinity over large areas. But in one final experiment, the authors also found evidence for an even wider significance. They found that SPIRRIG's BEACH domain bound to both mammalian and yeast forms of DCP1, and that a human BEACH-containing protein bound to the Arabidopsis DCP1. Taken together, these results suggest that BEACH domain proteins likely play a similar RNA-sequestering role across the spectrum of eukaryotic organisms, and that understanding the details of this stress response in one organism is likely to shed light on similar responses in many others.

The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization

Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein–protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

The BEACH is hot: a LYST of emerging roles for BEACH-domain containing proteins in human disease.

BEACH (named after 'Beige and Chediak-Higashi') is a conserved ∼280 residue domain, present in nine human BEACH domain containing proteins (BDCPs). Most BDCPs are large, containing a PH-like domain for membrane association preceding their BEACH domain, and containing WD40 and other domains for ligand binding. Recent studies found that mutations in individual BDCPs cause several human diseases. BDCP alterations affect lysosome size (LYST and NSMAF), apoptosis (NSMAF), autophagy (LYST, WDFY3, LRBA), granule size (LYST, NBEAL2, NBEA) or synapse formation (NBEA). However, the roles of each BDCP in these membrane events remain controversial. After reviewing studies on individual BDCPs, we propose a unifying hypothesis that BDCPs act as scaffolding proteins that facilitate membrane events, including both fission and fusion, determined by their binding partners. BDCPs may also bind each other, enabling fusion or fission of vesicles that are not necessarily of the same type. Such mechanisms explain why different BDCPs may have roles in autophagy; each BDCP is specific for the cell type or the cargo, but not necessarily specific for attaching to the autophagosome. Further elucidation of these mechanisms, preferably carrying out the same experiment on multiple BDCPs, and possibly using patients' cells, may identify potential targets for therapy.

Sphingolipid metabolism modulates motor neuron degeneration in Drosophila

We are interested in the involvement of sphingolipids in neurodegeneration in Drosophila. To look at this, we study animals deficient in Blue cheese, a BEACH domain protein involved in autophagy and lysosomal transport, and highly expressed in the nervous system. Blue cheese mutants exhibit shortened life span, extensive neuronal loss and accumulation of ubiquitinated proteins in the brain (Finley et al, 2003). The role of BEACH domain in the FAN protein, which triggers neutral sphingomyelinase activity (Segui et al, 1999), and the involvement of sphingolipids in various human neurodegenerative conditions (Cutler et al, 2002, 2004; Han et al, 2004), led us to hypothesize that blue cheese mutation could lead to degeneration by altering sphingolipid balance. We introduced mutations in the sphingolipid‐biosynthetic pathway into the blue cheese background to look for modification of the phenotype (motorneuron count). Lace (SPT) and slab (CDase) mutants and overexpressors in the background of the blue cheese mutation indeed modulated neuronal survival, in a way that suggests that ceramide availability in the brain is critical. Mass spectrometric quantification of sphingolipid levels in mutant larval brains show dysregulation of ceramides and changes in activation of putative ceramide‐regulated MAPK signaling pathway targets P‐Akt, P‐JNK and P‐MKK4, which may affect neuronal survival. Based on these sphingolipid changes and genetic interactions, we propose a possible mechanism whereby blue cheese may regulate sphingomyelinase and SPT, and act synergistically with ceramidase.Research Grant: Biomedical Research Council (BMRC), A‐star, Singapore

Neurobeachin regulates neurotransmitter receptor trafficking to synapses.

The surface density of neurotransmitter receptors at synapses is a key determinant of synaptic efficacy. Synaptic receptor accumulation is regulated by the transport, postsynaptic anchoring, and turnover of receptors, involving multiple trafficking, sorting, motor, and scaffold proteins. We found that neurons lacking the BEACH (beige-Chediak/Higashi) domain protein Neurobeachin (Nbea) had strongly reduced synaptic responses caused by a reduction in surface levels of glutamate and GABA(A) receptors. In the absence of Nbea, immature AMPA receptors accumulated early in the biosynthetic pathway, and mature N-methyl-d-aspartate, kainate, and GABA(A) receptors did not reach the synapse, whereas maturation and surface expression of other membrane proteins, synapse formation, and presynaptic function were unaffected. These data show that Nbea regulates synaptic transmission under basal conditions by targeting neurotransmitter receptors to synapses.