Signaling by K63-linked polyubiquitin in autophagy-related vesicular trafficking

2019 Fall.Includes bibliographical references.In eukaryotic cells, endocytosis and autophagy represent fundamental pathways that contribute to cell homeostasis. Intersection of the endocytic and autophagosomal trafficking routes has been previously recognized, as both systems share specific compartments contributing to cargo degradation via the lysosome, and both use an identical subset effectors involved in vesicular trafficking and membrane fusion. By helping to target proteins to the lysosome, ubiquitin (Ub) also acts as a common factor in both pathways. The relative contributions of endocytic and autophagy pathways to degradation of ubiquitinated cargo are mostly unknown. A critical unanswered question is how ubiquitinated cargo is targeted to a particular degradative pathway. Ub can be conjugated to other proteins as a single Ub or chains of multiple Ub (polyUb). Many types of polyUb, distinguished by different Ub–Ub linkages, are present in cells, where they are thought to be recognized by decoders (i.e., receptors or adaptors) to signal various biological processes. K63-polyUb has been implicated in endocytosis of cell surface receptors, budding of viral proteins, and autophagy of damaged organelles and invading pathogens. However, the extent and details of the relationship between K63-polyUb and lysosomal targeting via either endocytosis or autophagy are poorly understood. These deficiencies are due in part to the lack of tools to study Ub-dependent signaling. We have previously developed a genetically-encoded specific K63-polyUb sensor, "Vx3," to facilitate identification, localization, and functional characterization of signaling by K63-polyUb in vivo [Sims et al., Nat. Methods (2012)]. When Vx3 is expressed in cells, it localizes to cytoplasmic foci that colocalize with the autophagic transmembrane protein ATG9A, the autophagic cargo receptor p62 (SQSTM1), which preferentially bind K63-polyUb, and late endosomal/lysosomal markers. Proteins identified by mass spectrometry and likely modified by K63-polyUb appear to be transmembrane proteins known to recycle between endosomes and plasma membrane, such as transferrin receptor (TfR) and major histocompatibility complex I (MHC-I). Here, we show that K63-polyUb modifies endocytic transmembrane proteins to signal their delivery to the lysosomes. Fluorescence and electron microscopy data revealed that Vx3 accumulates K63-polyUb-decorated vesicles into cytoplasmic foci at an intermediate stage of the transmembrane protein degradation pathway; these foci include components of both endocytic and autophagy membranes. We observed that K63-polyUb-decorated vesicles co-clustering with ATG9A are found juxtaposed to endolysosomes, consistent with the idea of fusion block imposed by Vx3. By using a system to inducibly disable Vx3 inhibition, we tracked delivery of K63-polyubiquitinated cargo to the highly acidic and degradative lysosomal compartments. We also report that ESCRT dysfunction leads to accumulation Vx3 foci, indicating an augmentation of K63-polyubiquinated cargo destined to lysosomal degradation via the multivesicular late endosomes. Despite the established role for endocytosis in cargo sorting and recycling, there is evidence that autophagy may also play a role in regulating these events. By using Vx3 to inhibit autophagy-related membrane trafficking, our study also reveal a functional crosstalk between the Ub-dependent sorting of endocytic cargo into multivesicular late endosomes and the retromer-mediated retrieval of transmembrane proteins from maturing endosomes

Preventable Cases of Oral Anticoagulant-Induced Bleeding: Data From the Spontaneous Reporting System

Background: Despite the risk of bleeding is a well-known adverse effect of oral anticoagulants, there is scarce evidence on the preventability of oral anticoagulant-induced bleedings. Therefore, we investigated the potential risk factors related to preventable cases of oral anticoagulant-induced bleedings. Methods: We performed a study using Individual Case Safety Reports (ICSRs) with an oral anticoagulant as suspected drug among those reported through the spontaneous reporting system of Campania Region from 1 July 2012 to 31 December 2017. The P-method was used for the preventability assessment of all cases of bleeding. Results: In total, 58 cases out of 253 (22.9%) were preventable, and the most reported suspected drug was an indirect oral anticoagulant (warfarin). Sixty-eight critical criteria for preventability were identified, all related to healthcare professionals' practices. The most detected risk factor related to healthcare professionals' practices was the labeled drug-drug interaction for both direct and indirect oral anticoagulants. Conclusion: Our findings describe the most reported risk factors for preventability of oral anticoagulant-induced bleedings. These factors may be useful for targeting interventions to improve pharmacovigilance activities in our regional territory and to reduce the burden of medication errors and inappropriate prescription

The New Paradigms in Clinical Research: From Early Access Programs to the Novel Therapeutic Approaches for Unmet Medical Needs

Despite several innovative medicines gaining worldwide approval in recent years, there are still therapeutic areas for which unsatisfied therapeutic needs persist. For example, high unmet clinical need was observed in patients diagnosed with type 2 diabetes mellitus and hemophilia, as well as in specific age groups, such as the pediatric population. Given the urgent need to improve the therapy of clinical conditions for which unmet clinical need is established, clinical testing, and approval of new medicines are increasingly being carried out through accelerated authorization procedures. Starting from 1992, the Food and Drug Administration and the European Medicines Agency have supported the so-called Early Access Programs (EAPs). Such procedures, which can be based on incomplete clinical data, allow an accelerated marketing authorization for innovative medicines. The growth in pharmaceutical research has also resulted in the development of novel therapeutic approaches, such as biotech drugs and advanced therapy medicinal products, including new monoclonal antibodies for the treatment of asthma, antisense oligonucleotides for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy, and new anticancer drugs that act on genetic biomarkers rather than any specific type of cancer. Even though EAPs and novel therapeutic approaches have brought huge benefits for public health, their implementation is limited by several challenges, including the high risk of safety-related label changes for medicines authorized through the accelerated procedure, the high costs, and the reimbursement and access concerns. In this context, regulatory agencies should provide the best conditions for the implementation of the described new tools

Gender Differences in Outpatient Pediatric Drug Utilization: A Cohort Study From Southern Italy

Objective: The aim of this retrospective population-based cohort study is to in-depth investigate gender-specific drug utilization pattern in pediatric outpatient population.Methods: By using a large administrative database of the Local Health Unit of Caserta (Southern Italy), a pediatric cohort from the birth to 18 years was observed over 6 years (from 1st January 2010 to 31st December 2015). Yearly prevalence of drug use per 100 inhabitants as well as the median number of prescriptions was stratifying by gender. Prevalence of acute and recurrent use of the most frequently used active substances was calculated for the year 2015.Results: A decreasing trend in prevalence of drug use (−3.2%, with a reduction of median number of drugs dispensed) was observed in children for both sexes, from 2010 to 2015. In 2015, the drug classes most commonly used among children of any age were modestly but consistently prescribed more to males than to females: systemic anti-infective drugs (M = 43.5%; F = 42.3%), respiratory tract drugs (M = 29.0%; F = 26.1%), and hormones (M = 13.1%; F = 11.3%). Irrespective of gender, beclomethasone was the most utilized active substance in the first 2 years of life, while thereafter amoxicillin/clavulanate in combination.Conclusions: In a large population of pediatric outpatients no major difference was seen between genders, although commonly used drug classes; in particular, antibiotics, respiratory tract drugs and Hormones with corticosteroids for systemic use prescribed modestly but consistently to larger extent in males than females

Targeting prolyl-isomerase Pin1 prevents mitochondrial oxidative stress and vascular dysfunction: insights in patients with diabetes

The present study demonstrates that Pin1 is a common activator of key pathways involved in diabetic vascular disease in different experimental settings including primary human endothelial cells, knockout mice, and diabetic patients. Gene silencing and genetic disruption of Pin1 prevent hyperglycaemia-induced mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Moreover, we have translated our findings to diabetic patients. In line with our experimental observations, Pin1 up-regulation is associated with impaired flow-mediated dilation, increased oxidative stress, and plasma levels of adhesion molecules. In perspective, these findings may provide the rationale for mechanism-based therapeutic strategies in patients with diabete

Blood Cell-Bound C4d as a Marker of Complement Activation in Patients With the Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against phospholipid-binding proteins (aPL), such as beta2glycoprotein I (β2GPI). Complement is involved in APS animal models and complement deposits have been documented in placenta and thrombotic vessels despite normal serum levels. Analysis of circulating blood cells coated with C4d displays higher sensitivity than the conventional assays that measure soluble native complement components and their unstable activation products in systemic lupus erythematosus (SLE). As C4d-coated blood cell count has been reported to be more sensitive than serum levels of complement components and their activation products in systemic lupus erythematosus (SLE) patients, we decided to evaluate the percentage of C4d positive B lymphocytes (BC4d), erythrocytes (EC4d), and platelets (PC4d) in primary APS patients and asymptomatic aPL positive carriers as marker of complement activation in APS. We assessed by flow cytometry the percentages of BC4d, EC4d, and PC4d in primary APS (PAPS; n. 23), 8 asymptomatic aPL positive carriers, 11 APS-associated SLE (SAPS), 17 aPL positive SLE, 16 aPL negative SLE, 8 aPL negative patients with previous thrombosis, 11 immune thrombocytopenia (ITP) patients, and 26 healthy subjects. In addition, we used an in vitro model to evaluate the ability of a monoclonal anti-β2GPI antibody (MBB2) to bind to normal resting or activated platelets and fix complement. EC4d and PC4d percentages were significantly higher in PAPS and aPL carriers as well as aPL positive SLE and SAPS than in aPL negative controls. The highest values were found in PAPS and in SAPS. The EC4d and PC4d percentages were significantly correlated with serum C3/C4 and anti-β2GPI/anti-cardiolipin IgG. In vitro studies showed that MBB2 bound to activated platelets only and induced C4d deposition. The detection of the activation product C4d on circulating erythrocytes and platelets supports the role of complement activation in APS. Complement may represent a new therapeutic target for better treatment and prevention of disability of APS patients

CONSERVATION AND REHABILITATION TO MUSEUM OF LAURINI PALACE IN TITO, POTENZA, ITALY

The Palace owned by the Laurini family is an important building located in the historic center of the ancient city of Tito, a few kilometers from Potenza, in the heart of Southern Italy, built between the end of the 18th century and the end of the 19th century. It was built in three phases and three factory bodies joined together at different levels (up to five), with a very irregular volume both in plan and in section and a large sloping garden with a surface of over 3,800 m2. Thanks to the attention and care of the property, the building has fully conserved its original construction, and today appears as a living manual of the architecture of its time. This also and above all because the owners refused to apply the simplistic and invasive recipes practiced after the 1980 earthquake, which led to the almost complete destruction of the historical heritage of Campania and Basilicata. The Palace preserves untidy stone wall; the vaults in stone, barrel and cross-shaped (one also lathwork type, on the top floor); the original stairs; the floors in chestnut wood, the roofs with Palladian trusses and bent tiles; the frames of doors and windows, some very ornate, and the original wooden fixtures; railings and gratins in molded cast iron and wrought iron; the pavements in colored and decorated cement grit stones; the original plasters and colorings. The Notary Laurini intends to dedicate the building to the enhancement of the thought and work of Lorenzo Ostuni, personality of Tito internationally known, artist and scholar of symbols, who died a few years ago. And to this end it has set up a Foundation to which it has given the Palace, to make it become the seat of a Museum of Symbols and related study activities. The research carried out has determined the methods through which, in respect of the historic-artistic characteristics of the building, it reaches its anti-seismic adaptation, its re-functionalization, the elimination of humidity pathologies, also achieving a marked improvement in its energy performance and sustainability

Swarming and poly-gamma-glutamate synthesis depend on the synergic action of SwrAA and DegU in Bacillus subtilis

Isogenic populations of Bacillus subtilis are multicellular communities consisting of distinct differentiated cell types and the two component system DegS-DegU has a central role in the control of single cell fates. The level of DegU phosphorylation attained inside each cell is one of the main parameters used to select specific cell fates but is not the only parameter that triggers a particular genetic programme. In laboratory strains, cooperativity between DegU and SwrAA has been observed in motility: a wild-type copy of the gene (swrAA+) confers a full swimming capacity and the ability to swarm on semi-solid surfaces when DegU is present but it does not show any motility advantage if the two component system DegS/U is deleted (1). We will show that the synergic action of DegU and SwrAA is a conserved “modus operandi” since it is also observed in the production of poly-gamma-glutamic acid (gamma-PGA), an extracellular anionic polymer composed of thousands of glutamic acid residues linked by gamma-glutamyl bonds, produced and secreted by Bacilli. Domesticated B. subtilis strains synthesize gamma-PGA if they carry a degQH (2) or a degU32(Hy) / degS200(Hy) mutation and the wild type swrAA allele. These data indicate that the activation of the biosynthetic pgs operon is dependent on the co-presence of a high level of DegU~P and SwrAA. The presence of either SwrAA or DegU~P alone has only a marginal effect on gamma-PGA production and pgs operon transcription. The effect of SwrAA and DegU~P is cooperative rather than additive. Motility is not involved in gamma-PGA production since a sigD null mutation or a large deletion in the main flagellar operon (fla/che) do not affect gamma-PGA synthesis in degU32(Hy) swrAA+ strains. Moreover, a fla/che promoter up-mutation, that allows swarming and full swimming motilities in a degU32(Hy) swrAA- strain, does not confer the ability to produce gamma-PGA. Activation of gamma-PGA synthesis is therefore a motility-independent phenotype in which SwrAA and DegU~P display a cooperative effect. 1) Calvio et al., 2008. J Bacteriol 190:5720-28. 2) Stanley NR and Lazazzera BA, 2005. Mol Microbiol 57:1143-