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Glycine N-degron regulation revealed For more than 30 years, N-terminal sequences have been known to influence protein stability, but additional features of these N-end rule, or N-degron, pathways continue to be uncovered. Timms et al. used a global protein stability (GPS) technology to take a broader look at these pathways in human cells. Unexpectedly, glycine exposed at the N terminus could act as a potent degron; proteins bearing N-terminal glycine were targeted for proteasomal degradation by two Cullin-RING E3 ubiquitin ligases through the substrate adaptors ZYG11B and ZER1. This pathway may be important, for example, to degrade proteins that fail to localize properly to cellular membranes and to destroy protein fragments generated during cell death. Science , this issue p. eaaw4912

Summary The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFs complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.

Specific cellular fates and functions depend on differential gene expression, which occurs primarily at the transcriptional level, controlled by complex regulatory networks of transcription factors. Transcription factors act through combinatorial interactions with other transcription factors, co-factors and chromatin-remodelling proteins. We present a study of 459 Drosophila melanogaster transcription related factors, defining protein-protein interactions using a co-affinity purification mass spectrometry methodology, representing approximately half of the established catalogue of transcription factors. We probe this network in vivo, demonstrating functional interactions for many interacting proteins testing the predictive value for our data set. Building on these analyses, we combine regulatory network inference models with physical interactions to define an integrated network, connecting combinatorial transcription factor protein interactions to the transcriptional regulatory network of the cell. We use this integrated network as a tool to connect the functional network of genetic modifiers related to mastermind, a transcriptional co-factor of the Notch pathway.

Exudative retinopathy may be a manifestation of a variety of isolated ocular or systemic diseases in children. We report the case of a teenager with dyskeratosis congenita who developed a unilateral late exudative retinopathy after having previous laser treatment for threshold retinopathy of prematurity as an infant.

SummaryDetermining the composition of protein complexes is an essential step toward understanding the cell as an integrated system. Using coaffinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly 5,000 individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a statistical framework designed to define individual protein-protein interactions, led to the generation of a Drosophila protein interaction map (DPiM) encompassing 556 protein complexes. The high quality of the DPiM and its usefulness as a paradigm for metazoan proteomes are apparent from the recovery of many known complexes, significant enrichment for shared functional attributes, and validation in human cells. The DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. The DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution.

Connexin 43 knockout (Cx43 KO) mice exhibit conotruncal malformations and coronary artery defects. We observed epicardial blisters in the Cx43 KO hearts that suggest defects in epicardial epithelial-mesenchymal transformation (EMT), a process that generates coronary vascular progenitors. Analysis using a three-dimensional collagen gel invasion assay showed that Cx43 KO epicardial cells are less invasive and that, unlike wild-type epicardial cells, they fail to organize into thin vessel-like projections. Examination of Cx43 KO hearts using Wt1 as an epicardial marker revealed a disorganized pattern of epicardial cell infiltration. Time-lapse imaging and motion analysis using epicardial explants showed a defect in directional cell migration. This was associated with changes in the actin/tubulin cytoskeleton. A defect in cell polarity was indicated by a failure of the microtubule-organizing center to align with the direction of cell migration. Forced expression of Cx43 constructs in epicardial explants showed the Cx43 tubulin-binding domain is required for Cx43 modulation of cell polarity and cell motility. Pecam staining revealed early defects in remodeling of the primitive coronary vascular plexuses in the Cx43 KO heart. Together, these findings suggest an early defect in coronary vascular development arising from a global perturbation of the cytoarchitecture of the cell. Consistent with this, we found aberrant myocardialization of the outflow tract, a process also known to be EMT dependent. Together, these findings suggest cardiac defects in the Cx43 KO mice arise from the disruption of cell polarity, a process that may be dependent on Cx43-tubulin interactions.

Purpose To describe the anatomic and visual outcomes of patients in whom noninfectious endophthalmitis developed after injection of intravitreal triamcinolone acetonide. Design Retrospective case series. Methods Ophthalmologic evaluations of patients in whom noninfectious endophthalmitis developed after intravitreal triamcinolone took place on the day of injection, at the time of presentation of noninfectious endophthalmitis, at the time of clearance of inflammation, and on follow-up examination. Seventeen eyes of 17 patients were identified from 2 institutions. Noninfectious endophthalmitis was identified based on history of visual loss immediately or soon after injection, lack of ocular pain, hypopyon, anterior or vitreous inflammation, and triamcinolone crystals present in the anterior or posterior chambers. Main outcome measures were Snellen visual acuity (VA) and mean foveal thickness by optical coherence tomography. Results Mean VA and mean foveal thickness on the day of injection of intravitreal triamcinolone were 20/132 (logarithm of the minimum angle of resolution [logMAR], 0.82 ± 0.45) and 432 ± 118 μm, respectively. Mean VA at time of noninfectious endophthalmitis (mean, 1.9 days after injection) was 20/4444 (logMAR, 2.35 ± 0.98). At last follow-up (mean, 57.6 days), VA and mean foveal thickness were 20/56 (logMAR, 0.44 ± 0.30) and 301 ± 71 μm, respectively. Conclusions VA and mean foveal thickness in all patients with noninfectious endophthalmitis after intravitreal triamcinolone improved to better than preinjection levels in this series. At last follow-up, no patient had sustained visual loss from noninfectious endophthalmitis. Noninfectious endophthalmitis after intravitreal triamcinolone may not exclude good visual and anatomic prognoses.

(Cell 146, 359–371; August 5, 2011)In this paper, we described the directed conversion of skin fibroblasts from unaffected individuals or familial Alzheimer’s disease patients into human induced neuronal cells. We also presented molecular analyses of Alzheimer’s-associated markers in these cells. Dr. Ryousuke Fujita, who was specifically and only responsible for the molecular analyses of Alzheimer’s-associated pathology, has acknowledged inappropriately manipulating image panels and data points, as well as misrepresenting the number of repeats performed, in the experiments presented in Figures 6 and 7 of the paper (and corresponding Figures S5 and S6). We are in the process of repeating these analyses. Given these issues, we believe that the most appropriate course of action is to retract the paper. We deeply regret this circumstance and apologize to the community.

Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation.

North Carolina macular dystrophy is characterized by nonprogressive atrophy of the choroid, choriocapillaris, and the retinal pigment epithelial layer. 1,2 The characteristic retinal findings, ranging from scattered drusen to a posterior staphyloma, have been reported as early as infancy and usually reach their greatest magnitude in the second decade of life. 3 Herein, we describe a case of a 3-year-old boy with a documented family history of North Carolina macular dystrophy who presented with a subfoveal choroidal neovascular membrane in addition to the macular dystrophic changes.

Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aβ, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.