Hippo pathway activity influences liver cell fate.

The Hippo-signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level. We also identify the NOTCH-signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration.

Lifelong multilineage contribution by embryonic-born blood progenitors.

Haematopoietic stem cells (HSCs) arise in the embryo from the arterial endothelium through a process known as the endothelial-to-haematopoietic transition (EHT)1-4. This process generates hundreds of blood progenitors, of which a fraction go on to become definitive HSCs. It is generally thought that most adult blood is derived from those HSCs, but to what extent other progenitors contribute to adult haematopoiesis is not known. Here we use in situ barcoding and classical fate mapping to assess the developmental and clonal origins of adult blood in mice. Our analysis uncovers an early wave of progenitor specification-independent of traditional HSCs-that begins soon after EHT. These embryonic multipotent progenitors (eMPPs) predominantly drive haematopoiesis in the young adult, have a decreasing yet lifelong contribution over time and are the predominant source of lymphoid output. Putative eMPPs are specified within intra-arterial haematopoietic clusters and represent one fate of the earliest haematopoietic progenitors. Altogether, our results reveal functional heterogeneity during the definitive wave that leads to distinct sources of adult blood.

BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition.

BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 is a 0.5-MDa protein associated with multiple functions in Arabidopsis (Arabidopsis thaliana) signalling and development. However, the biochemical functions of BIG are unknown. We investigated a role for BIG in the Arg/N-degron pathways, in which substrate protein fate is influenced by the N-terminal (Nt) residue. We crossed a big loss-of-function allele to two N-degron pathway E3 ligase mutants, proteolysis6 (prt6) and prt1, and examined the stability of protein substrates. Stability of model substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants and the abundance of the PRT6 physiological substrates, HYPOXIA-RESPONSIVE ERF2 (HRE2) and VERNALIZATION2 (VRN2) was similarly increased in prt6 big double mutants. Hypoxia marker expression was enhanced in prt6 big double mutants; this constitutive response required arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated increased expression of multiple hypoxia-responsive genes in the double mutant relative to prt6, but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and broader processes in Arabidopsis.

Bioengineering secreted proteases convert divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors.

Secreted immune proteases Rcr3 (Required for Cladosporium resistance-3) and Pip1 (Phytophthora- inhibited protease-1) of tomato (Solanum lycopersicum) are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signalling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues, and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers.

The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54-61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54-61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21-28 residues from the activated αBΔ54-61 (to get αBΔ21-28, Δ54-61) on the structure-function of recombinant αBΔ21-28, Δ54-61. The αBΔ21-28, Δ54-61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21-28, ∆54-61 was found to prevent ß-amyloid (Aß1-42) fibril formation in vitro and suppressed Aß1-42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54-61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54-61 in oxidatively stressed cells. Our study shows that the residues 21-28 and 54-61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21-28 residues further potentiates the activated αBΔ54-61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21-28, Δ54-61.

A Comprehensive Study of Gradient Conditions for Deep Proteome Discovery in a Complex Protein Matrix.

Bottom-up mass-spectrometry-based proteomics is a well-developed technology based on complex peptide mixtures from proteolytic cleavage of proteins and is widely applied in protein identification, characterization, and quantitation. A tims-ToF mass spectrometer is an excellent platform for bottom-up proteomics studies due to its rapid acquisition with high sensitivity. It remains challenging for bottom-up proteomics approaches to achieve 100% proteome coverage. Liquid chromatography (LC) is commonly used prior to mass spectrometry (MS) analysis to fractionate peptide mixtures, and the LC gradient can affect the peptide fractionation and proteome coverage. We investigated the effects of gradient type and time duration to find optimal gradient conditions. Five gradient types (linear, logarithm-like, exponent-like, stepwise, and step-linear), three different gradient lengths (22 min, 44 min, and 66 min), two sample loading amounts (100 ng and 200 ng), and two loading conditions (the use of trap column and no trap column) were studied. The effect of these chromatography variables on protein groups, peptides, and spectral counts using HeLa cell digests was explored. The results indicate that (1) a step-linear gradient performs best among the five gradient types studied; (2) the optimal gradient duration depends on protein sample loading amount; (3) the use of a trap column helps to enhance protein identification, especially low-abundance proteins; (4) MSFragger and PEAKS Studio have high similarity in protein group identification; (5) MSFragger identified more protein groups among the different gradient conditions compared to PEAKS Studio; and (6) combining results from both database search engines can expand identified protein groups by 9-11%.

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S-RNase activity.

In self-incompatible Solanaceae, the pistil protein S-RNase contributes to S-specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S-RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S-RNase in vitro. Here, we show that expressing a redox-inactive mutant, NaTrxhSS , suppresses both S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys155 -Cys185 disulphide bond of SC10 -Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S-RNase activity by NaTrxh helps to explain why S-RNase alone could be insufficient for pollen rejection.

Cutting the line: manipulation of plant immunity by bacterial type III effector proteases.

Pathogens and their hosts are engaged in an evolutionary arms race. Pathogen-derived effectors promote virulence by targeting components of a host's innate immune system, while hosts have evolved proteins that sense effectors and trigger a pathogen-specific immune response. Many bacterial effectors are translocated into host cells using type III secretion systems. Type III effector proteases irreversibly modify host proteins by cleavage of peptide bonds and are prevalent among both plant and animal bacterial pathogens. In plants, the study of model effector proteases has yielded important insights into the virulence mechanisms employed by pathogens to overcome their host's immune response, as well as into the mechanisms deployed by their hosts to detect these effector proteases and counteract their effects. In recent years, the study of a larger number of effector proteases, across a wider range of pathogens, has yielded novel insights into their functions and recognition. One key limitation that remains is the lack of methods to detect protease cleavage at the proteome-wide level. We review known substrates and mechanisms of plant pathogen type III effector proteases and compare their functions with those of known type III effector proteases of mammalian pathogens. Finally, we discuss approaches to uncover their function on a system-wide level.

Clinicopathological findings in patients with COVID-19-associated ischaemic enterocolitis.

AIMS: Coronavirus disease 2019 (COVID-19) has been recognised as a predominantly respiratory tract infection, but some patients manifest severe systemic symptoms/coagulation abnormalities. The aim of this study was to evaluate the impact of severe COVID-19 infection on the gastrointestinal tract. METHODS AND RESULTS: We examined clinicopathological findings in 28 resected ischaemic bowels from 22 patients with severe COVID-19. Most patients required intubation preoperatively and presented with acute decompensation shortly before surgery. D-dimer levels were markedly elevated in all measured cases (mean, 5394 ng/ml). Histologically, 25 cases (19 patients) showed evidence of acute ischaemia with necrosis. In this group, the most characteristic finding was the presence of small vessel fibrin thrombi (24 of 25 cases, 96%), which were numerous in 64% of cases. Patients with COVID-19 were significantly more likely than a control cohort of 35 non-COVID-19-associated acute ischaemic bowels to show isolated small intestine involvement (32% versus 6%, P < 0.001), small vessel fibrin thrombi (100% versus 43%, P < 0.001), submucosal vessels with fibrinous degeneration and perivascular neutrophils (90% versus 54%, P < 0.001), fibrin strands within submucosal vessels (58% versus 20%, P = 0.007), and histological evidence of pneumatosis (74% versus 34%, P = 0.010). Three cases in this cohort had histopathological findings normally seen in the setting of chronic ischaemia, notably prominent fibroblastic proliferation affecting the outer layer of the muscularis propria. CONCLUSIONS: Herein, we describe the histopathological findings in COVID-19-associated ischaemic bowels and postulate a relationship with the hypercoagulable state seen in patients with severe COVID-19 infection. Additional experience with these cases may further elucidate specific features or mechanisms of COVID-19-associated ischaemic enterocolitis.

Quantitative Proteomic Analysis of Chikungunya Virus-Infected Aedes aegypti Reveals Proteome Modulations Indicative of Persistent Infection.

The mosquito-borne chikungunya virus (CHIKV) poses a threat to human health in tropical countries throughout the world. The molecular interactions of CHIKV with its mosquito vector Aedes aegypti are not fully understood. Following oral acquisition of CHIKV via salinemeals, we analyzed changes in the proteome of Ae. aegypti in 12 h intervals by label-free quantification using a timsTOF Pro mass spectrometer. For each of the seven time points, between 2647 and 3167 proteins were identified among CHIKV-infected and noninfected mosquito samples, and fewer than 6% of those identified proteins were affected by the virus. Functional enrichment analysis revealed that the three pathways, Endocytosis, Oxidative phosphorylation, and Ribosome biogenesis, were enriched during CHIKV infection. On the other hand, three pathways of the cellular RNA machinery and five metabolism related pathways were significantly attenuated in the CHIKV-infected samples. Furthermore, proteins associated with cytoskeleton and vesicular transport, as well as various serine-type endopeptidases and metallo-proteinases, were modulated in the presence of CHIKV. Our study reveals biological pathways and novel proteins interacting with CHIKV in the mosquito. Overall, CHIKV infection caused minor changes to the mosquito proteome demonstrating a high level of adaption between the vector and the virus, essentially coexisting in a nonpathogenic relationship. The mass spectrometry data have been deposited to the MassIVE repository (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=abfd14f7015243c69854731998d55df1) with the data set identifier MSV000085115.

Label-Free Quantitative Phosphoproteomics Reveals Signaling Dynamics Involved in Embryogenic Competence Acquisition in Sugarcane.

In this study, a label-free quantitative phosphoproteomic analysis was performed to identify and quantify signaling events related to the acquisition of embryogenic competence in sugarcane. Embryogenic and nonembryogenic calli were compared at the multiplication phase, resulting in the identification of 163 phosphoproteins unique to embryogenic calli, 9 unique to nonembryogenic calli, and 51 upregulated and 40 downregulated in embryogenic calli compared to nonembryogenic calli. Data are available via ProteomeXchange with identifier PXD018054. Motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx], and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. The embryogenic-related phosphoproteins (those unique and upregulated in embryogenic calli) identified in the present study are related to abscisic acid-induced signaling and abiotic stress response; they include OSK3, ABF1, LEAs, and RD29Bs. On the other hand, the nonembryogenic-related phosphoproteins EDR1 and PP2Ac-2 are negative regulators of abscisic acid signaling, suggesting a relationship between phosphoproteins involved in the abscisic acid and stress responses in the acquisition of embryogenic competence. Moreover, embryogenic-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TOPLESS, and with RNA metabolism, including AGO1, DEAH5, SCL30, UB2C, and SR45, were identified to play potential roles in embryogenic competence. These results reveal novel phosphorylation sites for several proteins and identify potential candidate biomarkers for the acquisition of embryogenic competence in sugarcane.

Quantitative Proteomics Reveals Docosahexaenoic Acid-Mediated Neuroprotective Effects in Lipopolysaccharide-Stimulated Microglial Cells.

The high levels of docosahexaenoic acid (DHA) in cell membranes within the brain have led to a number of studies exploring its function. These studies have shown that DHA can reduce inflammatory responses in microglial cells. However, the method of action is poorly understood. Here, we report the effects of DHA on microglial cells stimulated with lipopolysaccharides (LPSs). Data were acquired using the parallel accumulation serial fragmentation method in a hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer. Over 2800 proteins are identified using label-free quantitative proteomics. Cells exposed to LPSs and/or DHA resulted in changes in cell morphology and expression of 49 proteins with differential abundance (greater than 1.5-fold change). The data provide details about pathways that are influenced in this system including the nuclear factor κ-light-chain-enhancer of the activated B cells (NF-κB) pathway. Western blots and enzyme-linked immunosorbent assay studies are used to help confirm the proteomic results. The MS data are available at ProteomeXchange.

Defects in long-chain 3-hydroxy acyl-CoA dehydrogenase lead to hepatocellular carcinoma: A novel etiology of hepatocellular carcinoma.

The incidence of both nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) have been increasing at an alarming rate. Little is known about NAFLD without cirrhosis as a risk for HCC. Here we report, for the first time, generation of a mouse model with a defect in long-chain 3-hydoxy acyl-CoA dehydrogenase (LCHAD). The LCHAD exon 15 deletion was embryonic lethal to the homozygous mice whereas heterozygous mice (HT) develop significant hepatic steatosis starting at young age (3 months old) and HCC at older age (>13 months old) without any evidence of fibrosis or cirrhosis. None of the wild-type (WT) mice developed steatosis and HCC (n = 39), whereas HT-LCHAD mice (n = 41) showed steatosis and ~20% (8/41) developed liver masses with histological features of HCC. Proteomic analysis of liver tissues from WT-mice and HT-mice with no signs of HCC was conducted. Proteins with significant changes in abundance were identified by mass spectrometry. Abundance of 24 proteins was significantly different (p < 0.01) between WT and HT-LCHAD mice. The proteins found to vary in abundance are associated with different cellular response processes ranging from intermediary metabolism of carbohydrate, protein and lipid to oxidative stress, signal transduction and the process of tumorigenesis. Protein expression pattern of the HT-LCHAD mouse liver indicates predisposition to HCC and suggests that impaired hepatic mitochondrial fatty acid oxidation plays an important role in the development and progression of HCC. To assess the implication of these studies in human disease, we demonstrated significant downregulation of HADHA transcripts in HCC patients.

Quantitative Proteomics of Zea mays Hybrids Exhibiting Different Levels of Heterosis.

Maize hybrids exhibiting heterosis (hybrid vigor) were generated from inbred parents with increasing genetic distance. B73 was used as the common female parent in crosses with N192 (low heterosis), MO17 (high-heterosis 1), and NC350 (high-heterosis 2). Total and mitochondria-enriched proteomes were analyzed from ear shoots of field-grown hybrids and their inbred parents. GeLCMS (1D SDS-PAGE fractionation, trypsin digestion, LTQ Orbitrap nano-RP-LC MS/MS) was used to analyze proteins, and spectral counting was used for quantitation. In total, 3,568 proteins were identified and quantified in hybrids including 2,489 in the mitochondria-enriched fraction and 2,162 in the total protein fraction. Sixty-one proteins were differentially abundant (p < 0.05) in one or both of the high-heterosis hybrids compared with the low-heterosis hybrid. For the total proteome, eight of these showed similar trends in abundance in both of the higher-heterosis hybrids. Nine proteins showed this heterosis-correlated pattern in the mitochondrial proteome, including a mitochondria-associated target of rapamycin (TOR) protein. Although differentially abundant proteins belong to various pathways, protein, and RNA metabolism, and stress responsive proteins were the major classes changed in response to increasing heterosis.

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization.

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways.

IMMUNOTAR - Integrative prioritization of cell surface targets for cancer immunotherapy.

Cancer remains a leading cause of mortality globally. Recent improvements in survival have been facilitated by the development of less toxic immunotherapies; however, identifying targets for immunotherapies remains a challenge in the field. To address this challenge, we developed IMMUNOTAR, a computational tool that systematically prioritizes and identifies candidate immunotherapeutic targets. IMMUNOTAR integrates user-provided RNA-sequencing or proteomics data with quantitative features extracted from publicly available databases based on predefined optimal immunotherapeutic target criteria and quantitatively prioritizes potential surface protein targets. We demonstrate the utility and flexibility of IMMUNOTAR using three distinct datasets, validating its effectiveness in identifying both known and new potential immunotherapeutic targets within the analyzed cancer phenotypes. Overall, IMMUNOTAR enables the compilation of data from multiple sources into a unified platform, allowing users to simultaneously evaluate surface proteins across diverse criteria. By streamlining target identification, IMMUNOTAR empowers researchers to efficiently allocate resources and accelerate immunotherapy development.

Surface and Global Proteome Analyses Identify ENPP1 and Other Surface Proteins as Actionable Immunotherapeutic Targets in Ewing Sarcoma.

PURPOSE: Ewing sarcoma is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. Although the survival rate of patients diagnosed with localized disease is approximately 70%, this decreases to approximately 30% for patients with metastatic disease and only approximately 10% for treatment-refractory disease, which have not changed for decades. Therefore, new therapeutic strategies are urgently needed for metastatic and refractory Ewing sarcoma. EXPERIMENTAL DESIGN: This study analyzed 19 unique Ewing sarcoma patient- or cell line-derived xenografts (from 14 primary and 5 metastatic specimens) using proteomics to identify surface proteins for potential immunotherapeutic targeting. Plasma membranes were enriched using density gradient ultracentrifugation and compared with a reference standard of 12 immortalized non-Ewing sarcoma cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model to complement the surfaceome data. All models were analyzed by Tandem Mass Tags-based mass spectrometry to quantify identified proteins. RESULTS: The surfaceome and global proteome analyses identified 1,131 and 1,030 annotated surface proteins, respectively. Among surface proteins identified, both approaches identified known Ewing sarcoma-associated proteins, including IL1RAP, CD99, STEAP1, and ADGRG2, and many new cell surface targets, including ENPP1 and CDH11. Robust staining of ENPP1 was demonstrated in Ewing sarcoma tumors compared with other childhood sarcomas and normal tissues. CONCLUSIONS: Our comprehensive proteomic characterization of the Ewing sarcoma surfaceome provides a rich resource of surface-expressed proteins in Ewing sarcoma. This dataset provides the preclinical justification for exploration of targets such as ENPP1 for potential immunotherapeutic application in Ewing sarcoma. See related commentary by Bailey, p. 934.

The αA66-80 peptide interacts with soluble α-crystallin and induces its aggregation and precipitation: a contribution to age-related cataract formation.

Formation of protein aggregates in the aging eye lens has been shown to correlate with progressive accumulation of specific low-molecular weight (LMW) peptides derived from crystallins. Prominent among the LMW fragments is αA66-80, a peptide derived from αA-crystallin and present at higher concentrations in the water-insoluble nuclear fractions of the aging lens. The αA66-80 peptide has amyloid-like properties and preferentially insolubilizes α-crystallin from soluble lens fractions. However, the specific interactions and mechanisms by which the peptide induces α-crystallin aggregation have not been delineated. To gain insight into the mechanisms of peptide-induced aggregation, we investigated the interactions of the peptide with α-crystallin by various biochemical approaches. The peptide weakens α-crystallin chaperone ability and drastically promotes α-crystallin aggregation via the formation of insoluble peptide-protein complexes through transient intermediates. 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid studies suggest that the peptide induces changes in the hydrophobicity of α-crystallin that could trigger the formation and growth of aggregates. The peptide-α-crystallin aggregates were found to be resistant to dissociation by high ionic strengths, whereas guanidinium hydrochloride and urea were effective dissociating agents. We conclude that the αA66-80 peptide forms a hydrophobically driven, stable complex with α-crystallin and reduces its solubility. Using isotope-labeled chemical cross-linking and mass spectrometry, we show that the peptide binds to multiple sites, including the chaperone site, the C-terminal extension, and subunit interaction sites in αB-crystallin, which may explain the antichaperone property of the peptide and the consequential age-related accumulation of aggregated proteins. Thus, the α-crystallin-derived peptide could play a role in the pathogenesis of cataract formation in the aging lens.