FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

Local and global motion preferences in descending neurons of the fly

For a moving animal, optic flow is an important source of information about its ego-motion. In flies, the processing of optic flow is performed by motion sensitive tangential cells in the lobula plate. Amongst them, cells of the vertical system (VS cells) have receptive fields with similarities to optic flows generated during rotations around different body axes. Their output signals are further processed by pre-motor descending neurons. Here, we investigate the local motion preferences of two descending neurons called descending neurons of the ocellar and vertical system (DNOVS1 and DNOVS2). Using an LED arena subtending 240° × 95° of visual space, we mapped the receptive fields of DNOVS1 and DNOVS2 as well as those of their presynaptic elements, i.e. VS cells 1–10 and V2. The receptive field of DNOVS1 can be predicted in detail from the receptive fields of those VS cells that are most strongly coupled to the cell. The receptive field of DNOVS2 is a combination of V2 and VS cells receptive fields. Predicting the global motion preferences from the receptive field revealed a linear spatial integration in DNOVS1 and a superlinear spatial integration in DNOVS2. In addition, the superlinear integration of V2 output is necessary for DNOVS2 to differentiate between a roll rotation and a lift translation of the fly.     

Naturally Presented Peptides on Major Histocompatibility Complex I and II Molecules Eluted from Central Nervous System of Multiple Sclerosis Patients

Tandem mass spectrometry was used to identify naturally processed peptides bound to major histocompatibility complex (MHC) I and MHC II molecules in central nervous system (CNS) of eight patients with multiple sclerosis (MS). MHC molecules were purified from autopsy CNS material by immunoaffinity chromatography with monoclonal antibody directed against HLA-A, -B, -C, and -DR. Subsequently peptides were separated by reversed-phase HPLC and analyzed by mass spectrometry. Database searches revealed 118 amino acid sequences from self-proteins eluted from MHC I molecules and 191 from MHC II molecules, corresponding to 174 identified source proteins. These sequences define previously known and potentially novel autoantigens in MS possibly involved in disease induction and antigen spreading. Taken together, we have initiated the characterization of the CNS-expressed MHC ligandome in CNS diseases and were able to demonstrate the presentation of naturally processed myelin basic protein peptides in the brain of MS patients.T cells recognize antigen bound to MHC¹ molecules (1). CD4 as well as CD8 T cells have been shown to play a pathogenic role in various autoimmune diseases (2). Pathogenic T cells infiltrate the target organs and locally secrete proinflammatory cytokines and chemokines leading to tissue inflammation and possibly subsequent tissue destruction (3–5). Local presentation of autoantigens by MHC molecules in the target tissue of the autoimmune attack, i.e. the central nervous system (CNS) in multiple sclerosis (MS) or the pancreas in diabetes, is therefore a prerequisite for local immune amplification (6). MS is an inflammatory and neurodegenerative disease of the CNS leading to myelin and axonal loss (7). There are different disease courses, i.e. relapsing-remitting, secondary chronic progressive, and primary progressive disease. Potential autoantigens in MS include myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). It is thought that T cells enter the CNS from the systemic circulation and that they are subsequently reactivated in the CNS on MHC I and MHC II molecules expressed on local antigen-presenting cells (APC) (8).To date, naturally presented HLA-bound peptides from patients with MS thus far have not been isolated and identified. So far, only circumstantial evidence exists for the local presentation of autoantigens such as MBP on MHC molecules in CNS (9). The aim of this study consisted of the characterization of the MHC-bound peptide repertoire derived from brains of patients with MS. Cutting edge technology combining HPLC and tandem mass spectrometry has recently allowed us to define peptides presented on APC from bronchoalveolar lavage from lungs of sarcoidosis patients (10). Applying a similar method on autopsy material of MS patients, for the first time we demonstrated local presentation of previously known and potential novel autoantigens in MS.     

The disordered amino-terminus of SIMPL interacts with members of the 70-kDa heat-shock protein family

The p65 coactivator SIMPL is a small protein that lacks any conserved domains of known function. To better understand regulation of SIMPL activity, we sought to identify novel SIMPL interacting proteins using mass spectrometry analysis of SIMPL containing complexes. Two members of the 70-kDa heat-shock protein family, Hsp70 and Hsc70, were identified as SIMPL binding proteins. Subsequent immunocomplexing assays confirmed this interaction and demonstrated that the amino-terminus of SIMPL is required for this interaction. Using a combination of amino acid composition analysis, PONDR VL-XT prediction, charge-hydropathy plots, and cumulative distribution functions, the amino-terminal region of both mouse and human SIMPL proteins was predicted to be intrinsically disordered. These data, taken together, suggest that Hsp70/Hsc70 bind the intrinsically disordered amino-terminal region of SIMPL to stabilize the protein and thereby regulate its activity. Understanding the regulation of SIMPL through its interaction with Hsp70/Hsc70 may serve as a novel means of modulating tumor necrosis factor alpha signaling.     

Targeted disruption of CD38 accelerates autoimmune diabetes in NOD/Lt mice by enhancing autoimmunity in an ADP-ribosyltransferase 2-dependent fashion

Ubiquitously expressed CD38 and T cell-expressed ADP-ribosyltransferase 2 (ART2) are ectoenzymes competing for NAD substrate. CD38 exerts pleiotropic actions in hemopoietic and nonhemopoietic compartments via effects on calcium mobilization. ART2 is an ADP-ribosyltransferase on naive CD4+ and CD8+ T cells. ART2-catalyzed ADP-ribosylation of the P2X7 purinoreceptor elicits apoptosis. Transfer of a genetically disrupted CD38 allele into the autoimmune diabetes-prone NOD/Lt background accelerated diabetes onset in both sexes, whereas transfer of a disrupted ART2 complex had no effect. However, the fact that the accelerated pathogenesis mediated by CD38 deficiency required ART2 activity was demonstrated by combining both ART2 and CD38 deficiencies. Reciprocal bone marrow reconstitution studies demonstrated accelerated diabetes only when CD38-deficient bone marrow was transferred into CD38-deficient recipients. Neither decreases in beta cell function nor viability were indicated. Rather, the balance between T-effectors and T-regulatory cells was disturbed in CD38-deficient but ART2-intact NOD mice. In these mice, significant reductions in total viable CD8+ T cells were observed. This was accompanied by an age-dependent increase in a diabetogenic CD8 clonotype. This in turn correlated with impaired T-regulatory development (10-fold reduction in Foxp3 mRNA expression). These changes were corrected when CD38 deficiency was combined with ART2 deficiency. Both ART2-deficient and CD38/ART2 combined deficient T cells were resistant to NAD-induced killing in vitro, whereas CD38-deficient but ART2-intact T cells showed increased sensitivity, particularly the CD4+ CD25+ subset. Unexpectedly, diabetes development in the combined CD38/ART2 stock was strongly suppressed, possibly through epistatic interactions between genes linked to the targeted CD38 on Chromosome 5 and the ART2 complex on Chromosome 7.     

Mitochondrial and nuclear sequence polymorphisms reveal geographic structuring in Amazonian populations of Echinococcus vogeli (Cestoda: Taeniidae)

To date, nothing is known about the genetic diversity of the Echinococcus neotropical species, Echinococcus vogeli and Echinococcus oligarthrus. Here we used mitochondrial and nuclear DNA sequence polymorphisms to uncover the genetic structure, transmission and history of E. vogeli in the Brazilian Amazon, based on a sample of 38 isolates obtained from human and wild animal hosts. We confirm that the parasite is partially synanthropic and show that its populations are diverse. Furthermore, significant geographical structuring is found, with western and eastern populations being genetically divergent.     

Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.     

The Autism Psychodynamic Evaluation of Changes (APEC) scale: A reliability and validity study on a newly developed standardized psychodynamic assessment for youth with Pervasive Developmental Disorders

The present study was designed to examine the reliability and validity of the Autism Psychodynamic Evaluation of Changes (APEC) scale, developed to assess the evolution in individuals with autism under treatment. The APEC scale focuses on the key role of impairment in body image construction, which requires cross-modal sensory integration through emotional communication with motor representations. Thus, the body image construction is associated simultaneously with spatial and temporal organization and allows the emergence of self- and others-representations. The use of the APEC scale, with its seven domains (expression of emotion in relationships, eye contact, body image, graphic productions, exploration of space and objects, time perception, and verbal language), underlines the importance in autistic disorder of anxieties related to body and spatial representations, and of impairment in the body ego construction which is closely linked to the emergence of individuation/separation processes. This study was conducted on 73 children and adolescents with autistic disorder. They were recruited in day care facilities where two caregivers independently gave their ratings based on their clinical observation on a daily basis during the same month. Analyses included assessing construct validity through correspondence analyses and inter-rater reliability using kappa coefficients. The APEC scale offers a reliable and validated psychodynamic assessment of interest for professionals (such as child psychiatrists, caregivers, therapists or teachers) and researchers working with children, adolescents and adults with autistic disorder, especially in the follow-up of their evolution. The APEC scale provides an approach at the interface of psychoanalysis and neuroscience, and is also of interest for clinical and developmental psychology. Using the APEC scale in a range of different practical and research settings will foster links between psychoanalytic perspectives and educational training for children with autistic disorder, and will contribute to the dialogue between psychoanalysis, neuroscience and psychology.