Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.Gag | receptor | ubiquitin | vesicle

Prolonged stimulation of the b2-adrenergic receptor (b2AR) leads to receptor ubiquitination and downregulation. Using a genomewide RNA interference screen, we identified arrestin domaincontaining 3 (ARRDC3) as a gene required for b2AR regulation. The ARRDC3 protein interacts with ubiquitin ligase neural precursor development downregulated protein 4 (NEDD4) through two conserved PPXY motifs and recruits NEDD4 to the activated receptor. The ARRDC3 protein also interacts and co-localizes with activated b2AR. Knockdown of ARRDC3 expression abolishes the association between NEDD4 and b2AR. Furthermore, functional inactivation of ARRDC3, either through small interfering RNA (siRNA)-mediated knockdown or overexpression of a mutant that does not interact with NEDD4, blocks receptor ubiquitination and degradation. Our results establish ARRDC3 as an essential adaptor for b2AR ubiquitination.

Ivermectin (IVM) is a broad-spectrum anthelmintic used in filariasis control programs. By binding to nematode glutamate-gated chloride channels (GluCls), IVM disrupts neurotransmission processes regulated by GluCl activity. IVM treatment of filarial infections is characterized by an initial dramatic drop in the levels of circulating microfilariae, followed by long-term suppression of their production, but the drug has little direct effect on microfilariae in culture at pharmacologically relevant concentrations. We localized Brugia malayi GluCl expression solely in a muscle structure that surrounds the microfilarial excretory-secretory (ES) vesicle, which suggests that protein release from the ES vesicle is regulated by GluCl activity. Consistent with this hypothesis, exposure to IVM in vitro decreased the amount of protein released from microfilariae. To better understand the scope of IVM effects on protein release by the parasite, three different expression patterns were identified from immunolocalization assays on a representative group of five microfilarial ES products. Patterns of expression suggest that the ES apparatus is the main source of regulated ES product release from microfilariae, as it is the only compartment that appears to be under neuromuscular control. Our results show that IVM treatment of microfilariae results in a marked reduction of protein release from the ES apparatus. Under in vivo conditions, the rapid microfilarial clearance induced by IVM treatment is proposed to result from suppression of the ability of the parasite to secrete proteins that enable evasion of the host immune system. filarial nematode | macrocyclic lactone | glutamate-gated chloride channels

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich’s ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein–protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.

In Friedreich’s ataxia (FRDA) patients, diminished frataxin (FXN) in sensory neurons is thought to yield the predominant pathology associated with disease. In this study, we demonstrate successful usage of RNA transcript therapy (RTT) as an exogenous human FXN supplementation strategy in vitro and in vivo, specifically to dorsal root ganglia (DRG). Initially, 293 T cells were transfected with codon optimized human FXN mRNA, which was translated to yield FXN protein. Importantly, FXN was rapidly processed into the mature functional form of FXN (mFXN). Next, FXN mRNA, in the form of lipid nanoparticles (LNPs), was administered intravenously in adult mice. Examination of liver homogenates demonstrated efficient FXN LNP uptake in hepatocytes and revealed that the mitochondrial maturation machinery had efficiently processed all FXN protein to mFXN in ~24 h in vivo. Remarkably, greater than 50% mFXN protein derived from LNPs was detected seven days after intravenous administration of FXN LNPs, suggesting that the half-life of mFXN in vivo exceeds one week. Moreover, when FXN LNPs were delivered by intrathecal administration, we detected recombinant human FXN protein in DRG. These observations provide the first demonstration that RTT can be used for the delivery of therapeutic mRNA to DRG.

Iron-sulfur clusters (ISC) are essential in all life forms and carry out many crucial cellular functions.The core machinery for de novo ISC biosynthesis, located in the mitochondria matrix, is a fiveprotein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich's ataxia (FRDA). Recently crystal structures depicting the inactive 3-and 4-way sub-complexes of the ISC biosynthesis machinery, lacking the key activator FXN, have been determined. Here, the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates for the first time in any organism the interactions of FXN with the component proteins. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure sheds light on how FXN facilitates ISC production through unlocking the zinc inhibition and stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and offers an explanation of how FRDA clinical mutations affect complex formation and FXN activation.

No abstract

The AP1 (activator protein 1) transcription factor, c-Jun, is an important regulator of cell proliferation, differentiation, survival, and death. Its activity is regulated both at the level of transcription and post-translationally through phosphorylation, sumoylation, and targeted degradation. The degradation of c-Jun by the ubiquitin proteasome pathway has been well established. Here, we report that POH1, a subunit of the 19 S proteasome lid with a recently described deubiquitinase activity, is a regulator of c-Jun. Ectopic expression of POH1 in HEK293 cells decreased the level of c-Jun ubiquitination, leading to significant accumulation of the protein and a corresponding increase in AP1-mediated gene expression. The stabilization also correlated with a redistribution of c-Jun in the nucleus. These effects were reduced by mutation of a cysteine residue in the Mpr1 pad1 N-terminal plus motif of POH1 (Cys-120) and appeared to be selective for c-Jun, because POH1 had no effect on other proteasomal substrates. Our results identify a novel mechanism of c-Jun regulation in mammalian cells.The 26 S proteasome is the principal site of controlled protein degradation in eukaryotic cells. The 2-to 2.5-MDa proteasome complex consists of a central core (20 S proteasome) and one or two multiprotein regulatory particles (RPs 2 ; 19 S proteasome), which are further organized into base and lid regions (1). The 19 S RP mediates the binding, deubiquitination, and unfolding of substrates into the 20 S core, where proteolysis takes place (2). One of the many cellular proteins targeted for degradation by the proteasome is the AP1 transcription factor, c-Jun (3). In addition to being the most potent transcriptional activator of its group, c-Jun is involved in a myriad of cellular activities, including tumorigenesis (4). Tight control of intracellular concentrations of active c-Jun is therefore required, a process that is achieved through rapid turnover by ubiquitination and degradation. Novel mechanisms for targeted ubiquitination of c-Jun were recently shown to be carried out by c-Jun-specific ubiquitin ligases, such as Itch (5) and SCF Fbw7 (6). Whereas much continues to be learned about mechanisms of c-Jun ubiquitination, little is known about the process of deubiquitination and its contribution to the overall regulation of c-Jun. Cellular deubiquitinases (DUBs) play an important role in controlled protein degradation (7); when bound to the proteasome they mediate the removal of ubiquitin chains as the substrate is being degraded, a step that facilitates substrate translocation into the 20 S chamber and allows for recycling of ubiquitin in the cell. DUBs also provide ubiquitin-editing activity, which can rescue particular substrates from being degraded by removing or trimming the degradation signal. A wide range of DUBs has been identified in eukaryotic cells, including enzymes with specificities for particular ubiquitinated substrates (8) but no specific c-Jun deubiquitinase activity has yet been reported.A number of studies hav...