Multicellular organisms are composed of tissues that have distinct functions requiring specialized proteomes. To define the proteome of a live animal with tissue and subcellular resolution, we adapted a localized proteomics technology for use in the multicellular model organism Caenorhabditis elegans. This approach couples tissue- and location-specific expression of the enzyme ascorbate peroxidase (APX), which enables proximity-based protein labeling in vivo, and quantitative proteomics to identify tissue- and subcellular-restricted proteomes. We identified and localized more than 3000 proteins from strains of C. elegans expressing APX in either the nucleus or cytoplasm of the intestine, epidermis, body wall muscle, or pharyngeal muscle. We also identified several hundred proteins that were specifically localized to one of the four tissues analyzed or specifically localized to the cytoplasm or the nucleus. This approach resulted in the identification both of proteins with previously characterized localizations and of those not known to localize to the nucleus or cytoplasm. Further, we confirmed the tissue- and subcellular-specific localization of a subset of identified proteins using green fluorescent protein tagging and fluorescence microscopy, validating our in vivo proximity-based proteomics technique. Together, these results demonstrate a new approach that enables the tissue- and subcellular-specific identification and quantification of proteins within a live animal.

A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical "retrochaperone" for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates.

Background

Trimodality therapy (TMT) with preoperative chemoradiation followed by surgical resection is used for locally-advanced non-small-cell lung cancer (LA-NSCLC). Traditionally, preoperative radiation doses ≤54 Gy are used due to concerns regarding excess morbidity, but little is known about outcomes and toxicities after TMT with intensity-modulated radiotherapy (IMRT) to higher doses.

Methods

A retrospective analysis of patients who received planned TMT with IMRT for LA-NSCLC at Brigham and Women's Hospital/Dana-Farber Cancer Institute between 2008 and 2017 was performed. Clinical and treatment characteristics, pathologic response, and surgical toxicity were assessed. Kaplan-Meier method and log-rank test was used for survival outcomes. Cox proportional-hazards regression was used for multivariable analysis.

Results

Forty-six patients received less than definitive doses of <60 Gy and 30 patients received definitive doses ≥60 Gy. Surgical outcomes, pathologic complete response, and postoperative toxicity did not differ significantly between the groups. With median follow-up of 3.6 years (range: 0.4-11.4), three-year locoregional recurrence-free survival (78.0% vs. 68.3%, p = 0.51) and overall survival (OS) (61.0% vs. 69.4%, p = 0.32) was not significantly different between patients receiving <60 Gy and ≥60 Gy, respectively. On multivariable analysis, older age, clinical stage, and length of hospital stay (LOS) >7 days were associated with OS.

Conclusions

With IMRT, there was no increased rate of surgical complications in patients receiving higher doses of radiation. Survival outcomes or LOS did not differ based on radiation dose, but increased LOS was associated with worse OS. Larger prospective studies are needed to further examine outcomes after IMRT in patients with LA-NSCLC receiving TMT.

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify ∼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.

Background

Patients with non-small-cell lung cancer (NSCLC) that is resistant to PD-1 and PD-L1 (PD[L]-1)-targeted therapy have poor outcomes. Studies suggest that radiotherapy could enhance antitumour immunity. Therefore, we investigated the potential benefit of PD-L1 (durvalumab) and CTLA-4 (tremelimumab) inhibition alone or combined with radiotherapy.

Methods

This open-label, multicentre, randomised, phase 2 trial was done by the National Cancer Institute Experimental Therapeutics Clinical Trials Network at 18 US sites. Patients aged 18 years or older with metastatic NSCLC, an Eastern Cooperative Oncology Group performance status of 0 or 1, and progression during previous PD(L)-1 therapy were eligible. They were randomly assigned (1:1:1) in a web-based system by the study statistician using a permuted block scheme (block sizes of three or six) without stratification to receive either durvalumab (1500 mg intravenously every 4 weeks for a maximum of 13 cycles) plus tremelimumab (75 mg intravenously every 4 weeks for a maximum of four cycles) alone or with low-dose (0·5 Gy delivered twice per day, repeated for 2 days during each of the first four cycles of therapy) or hypofractionated radiotherapy (24 Gy total delivered over three 8-Gy fractions during the first cycle only), 1 week after initial durvalumab-tremelimumab administration. Study treatment was continued until 1 year or until progression. The primary endpoint was overall response rate (best locally assessed confirmed response of a partial or complete response) and, along with safety, was analysed in patients who received at least one dose of study therapy. The trial is registered with ClinicalTrials.gov, NCT02888743, and is now complete.

Findings

Between Aug 24, 2017, and March 29, 2019, 90 patients were enrolled and randomly assigned, of whom 78 (26 per group) were treated. This trial was stopped due to futility assessed in an interim analysis. At a median follow-up of 12·4 months (IQR 7·8-15·1), there were no differences in overall response rates between the durvalumab-tremelimumab alone group (three [11·5%, 90% CI 1·2-21·8] of 26 patients) and the low-dose radiotherapy group (two [7·7%, 0·0-16·3] of 26 patients; p=0·64) or the hypofractionated radiotherapy group (three [11·5%, 1·2-21·8] of 26 patients; p=0·99). The most common grade 3-4 adverse events were dyspnoea (two [8%] in the durvalumab-tremelimumab alone group; three [12%] in the low-dose radiotherapy group; and three [12%] in the hypofractionated radiotherapy group) and hyponatraemia (one [4%] in the durvalumab-tremelimumab alone group vs two [8%] in the low-dose radiotherapy group vs three [12%] in the hypofractionated radiotherapy group). Treatment-related serious adverse events occurred in one (4%) patient in the durvalumab-tremelimumab alone group (maculopapular rash), five (19%) patients in the low-dose radiotherapy group (abdominal pain, diarrhoea, dyspnoea, hypokalemia, and respiratory failure), and four (15%) patients in the hypofractionated group (adrenal insufficiency, colitis, diarrhoea, and hyponatremia). In the low-dose radiotherapy group, there was one death from respiratory failure potentially related to study therapy.

Interpretation

Radiotherapy did not increase responses to combined PD-L1 plus CTLA-4 inhibition in patients with NSCLC resistant to PD(L)-1 therapy. However, PD-L1 plus CTLA-4 therapy could be a treatment option for some patients. Future studies should refine predictive biomarkers in this setting.

Funding

The US National Institutes of Health and the Dana-Farber Cancer Institute.