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Membrane proteomics, the large-scale analysis of membrane proteins, is often constrained by the difficulties of achieving fully resolvable separation and resistance to proteolysis, both of which could lead to low recovery and low identification rates of membrane proteins. Here, we introduce a novel integrated approach, GELFrEE Optimized FASP Technology (GOFAST) for large-scale and comprehensive membrane proteins analysis. Using an array of sample preparation techniques including gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), filter-aided sample preparation (FASP), and microwave-assisted on-filter enzymatic digestion, we identified 2?090 proteins from the membrane fraction of a leukemia cell line (K562). Of these, 37% are annotated as membrane proteins according to gene ontology analysis, resulting in the largest membrane proteome of leukemia cells reported to date. Our approach combines the advantages of GELFrEE high-loading capacity, gel-free separation, efficient depletion of detergents, and microwave-assisted on-filter digestion, minimizing sample losses and maximizing MS-detectable sequence coverage of individual proteins. In addition, this approach also shows great potential for the identification of alternative splicing products. [ABSTRACT FROM AUTHOR]

Two different dispersion methods were studied in order to obtain rhenium [188Re] sulfide suspension with different particle size distributions. The manufactured swirling device offers Suspension I with larger particles (55%>5 µm, 19%>10 µm). However, ultrasonication can only produce Suspension II with smaller particles (93%<5 µm, 0.3%>10 µm). Stability tests indicated that deposition appears after 6 and 15 minutes for Suspensions I and II, respectively. Radiochemical purity and particle size distribution did not change distinctively within 24 hours. So, both suspensions can be used in animal tests to find out the optimal particle size ranges for intra-articular injection. [ABSTRACT FROM AUTHOR]

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is poorly understood due to a lack of an animal model that recapitulates severe human disease. Here, we report a Syrian hamster model that develops progressive lethal pulmonary disease that closely mimics severe coronavirus disease 2019 (COVID-19). We evaluated host responses using a multi-omic, multiorgan approach to define proteome, phosphoproteome, and transcriptome changes. These data revealed both type I and type II interferon-stimulated gene and protein expression along with a progressive increase in chemokines, monocytes, and neutrophil-associated molecules throughout the course of infection that peaked in the later time points correlating with a rapidly developing diffuse alveolar destruction and pneumonia that persisted in the absence of active viral infection. Extrapulmonary proteome and phosphoproteome remodeling was detected in the heart and kidneys following viral infection. Together, our results provide a kinetic overview of multiorgan host responses to severe SARSCoV- 2 infection in vivo. [ABSTRACT FROM AUTHOR]

3-M primordial dwarfism is an inherited disease characterized by severe pre- and postnatal growth retardation and by mutually exclusive mutations in three genes, CUL7, OBSL1, and CCDC8. The mechanism underlying 3-M dwarfism is not clear. We showed here that CCDC8, derived from a retrotransposon Gag protein in placental mammals, exclusively localized on the plasma membrane and was phosphorylated by CK2 and GSK3. Phosphorylation of CCDC8 resulted in its binding first with OBSL1, and then CUL7, leading to the membrane assembly of the 3-M E3 ubiquitin ligase complex. We identified LL5β, a plasma membrane protein that regulates cell migration, as a substrate of 3-M ligase. Wnt inhibition of CCDC8 phosphorylation or patient-derived mutations in 3-M genes disrupted membrane localization of the 3-M complex and accumulated LL5β. Deletion of Ccdc8 in mice impaired trophoblast migration and placental development, resulting in intrauterine growth restriction and perinatal lethality. These results identified a mechanism regulating cell migration and placental development that underlies the development of 3-M dwarfism. [ABSTRACT FROM AUTHOR]

Data from the Encyclopedia of DNA Elements (ENCODE) project show over 9640 human genome loci classified as long noncoding RNAs (lncRNAs), yet only ∼100 have been deeply characterized to determine their role in the cell. To measure the protein-coding output from these RNAs, we jointly analyzed two recent data sets produced in the ENCODE project: tandem mass spectrometry (MS/MS) data mapping expressed peptides to their encoding genomic loci, and RNA-seq data generated by ENCODE in long polyA+ and polyA-- fractions in the cell lines K562 and GM12878. We used the machinelearning algorithm RuleFit3 to regress the peptide data against RNA expression data. The most important covariate for predicting translation was, surprisingly, the Cytosol polyA-- fraction in both cell lines. LncRNAs are ∼13-fold less likely to produce detectable peptides than similar mRNAs, indicating that ∼92% of GENCODE v7 lncRNAs are not translated in these two ENCODE cell lines. Intersecting 9640 lncRNA loci with 79,333 peptides yielded 85 unique peptides matching 69 lncRNAs. Most cases were due to a coding transcript misannotated as lncRNA. Two exceptions were an unprocessed pseudogene and a bona fide lncRNA gene, both with open reading frames (ORFs) compromised by upstream stop codons. All potentially translatable lncRNA ORFs had only a single peptide match, indicating low protein abundance and/or false-positive peptide matches. We conclude that with very few exceptions, ribosomes are able to distinguish coding from noncoding transcripts and, hence, that ectopic translation and cryptic mRNAs are rare in the human lncRNAome. [ABSTRACT FROM AUTHOR]

Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD 1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mappedto either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD! monomer formation, a necessary initiating step in SOD! aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nM rather than 212 nM at physiological SOD! concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD! monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. [ABSTRACT FROM AUTHOR]