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Recent advances in nucleic acid sequencing now permit rapid and genome-scale analysis of genetic variation and transcription, enabling population-scale studies of human biology, disease, and diverse organisms. Likewise, advances in mass spectrometry proteomics now permit highly sensitive and accurate studies of protein expression at the whole proteome-scale. However, most proteomic studies rely on consensus databases to match spectra to peptide and proteins sequences, and thus remain limited to the analysis of canonical protein sequences. Here, we develop ProteomeGenerator2 (PG2), based on the scalable and modular ProteomeGenerator framework. PG2 integrates genome and transcriptome sequencing to incorporate protein variants containing amino acid substitutions, insertions, and deletions, as well as non-canonical reading frames, exons, and other variants caused by genomic and transcriptomic variation. We benchmarked PG2 using synthetic data and genomic, transcriptomic, and proteomic analysis of human leukemia cells. PG2 can be integrated with current and emerging sequencing technologies, assemblers, variant callers, and mass spectral analysis algorithms, and is available open-source fromhttps://github.com/kentsisresearchgroup/ProteomeGenerator2.

Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and biotherapeutic target. To uncover the biologically relevant structure of sNS1, we extracted the native form of sNS1 from cells infected with either the DENV WT or T164S mutant whose appearance was associated with a dengue outbreak in Cuba. We determined the cryoEM structures of sNS1 and its complex with a monoclonal antibody/Fab and found that the major species of sNS1 is a 1:1 complex of the NS1 dimer embedded in a High Density Lipoprotein (HDL) particle. Cross-linking MS studies confirm NS1:ApoA1 dimer formation with most ApoA1 interaction sites mapped to the NS1 wing and hydrophobic domains. Our results shed fresh light on the molecular pathogenesis of dengue and may have broad implications for managing dengue infection.SummaryCryoEM structures of secreted dengue virus NS1 protein reveal dimers in complex with high-density lipoprotein.

Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for the distinct functions of Smc5/6 is poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 holo-complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that the Smc5/6 complex possesses several unique features, while sharing some architectural characteristics with other SMC complexes. In contrast to arm-folded structures of cohesin and condensin, Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5/Nse6 subcomplex, with the latter also serving as a linchpin connecting distal parts of the complex. Our 3.0-Å resolution cryoelectron microscopy structure of the Nse5/Nse6 core further reveals a clasped-hand topology and a dimeric interface important for cell growth. Finally, we provide evidence that Nse5/Nse6 uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative study identifies distinct structural features of the Smc5/6 complex and functional cooperation among its coevolved unique subunits.

Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for Smc5/6’s distinct functions is currently poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that while the complex shares a similar overall organization with other SMC complexes, it possesses several unique features. In contrast to the reported folded-arm structures of cohesin and condensin, our data suggest that Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5-Nse6 subcomplex. Further, we show that Nse5-Nse6 subcomplex adopts a novel structure with an extensive dimerization interface and multiple domains contacting other subunits of the Smc5/6 complex. We also provide evidence that the Nse5-Nse6 module uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative multi-scale study identifies distinct structural features of the Smc5/6 complex and functional cooperation amongst its co-evolved unique subunits.

Human and animal tissues are frequently fresh frozen for archival storage. These specimens can be used for proteomic analysis to analyze proteome composition. A critical step in these proteomic analyses is the extraction of proteins from fresh frozen samples, which can affect the sampling of the proteomes. Here, we compare three different methods for protein extraction from fresh frozen mouse heart tissue: 1) extraction using SDS buffer followed by FASP purification, 2) extraction using SDS buffer followed by S-Trap purification, and 3) extraction using a guanidine hydrochloride buffer followed by in-solution digestion. Based on bicinchoninic acid assay, all three methods display similar recovery of total protein content. However, proteomics-based analysis identified far fewer proteins from the extraction guanidine hydrochloride. SDS-FASP identifies as many proteins as the SDS-STrap method with good coverage of proteins from different cellular compartments.

Summary The nuclear lamina is a universal feature of metazoan nuclear envelopes (NEs) [1]. In mammalian cells, it appears as a 10–30 nm filamentous layer at the nuclear face of the inner nuclear membrane (INM) and is composed primarily of A- and B-type lamins, members of the intermediate filament family [2]. While providing structural integrity to the NE, the lamina also represents an important signaling and regulatory platform [3]. Two A-type lamin isoforms, lamins A and C (LaA and LaC), are expressed in most adult human cells. Encoded by a single gene, these proteins are largely identical, diverging only in their C-terminal tail domains. By contrast with that of LaC, the unique LaA tail undergoes extensive processing, including farnesylation and endo-proteolysis [4, 5]. However, functional differences between LaA and LaC are still unclear. Compounding this uncertainty, the structure of the lamina remains ill defined. In this study, we used BioID, an in vivo proximity-labeling method to identify differential interactors of A-type lamins [6]. One of these, Tpr, a nuclear pore complex (NPC) protein, is highlighted by its selective association with LaC. By employing superresolution microscopy, we demonstrate that this Tpr association is mirrored in enhanced interaction of LaC with NPCs. Further superresolution studies visualizing both endogenous A- and B-type lamins have allowed us to construct a nanometer-scale model of the mammalian nuclear lamina. Our data indicate that different A- and B-type lamin species assemble into separate filament networks that together form an extended composite structure at the nuclear periphery providing attachment sites for NPCs, thereby regulating their distribution.

SummaryAntimetabolites that affect nucleotide metabolism are frontline chemotherapy agents in several cancers and often successfully target one carbon metabolism. However, the precise mechanisms and resulting determinants of their therapeutic value are unknown. We show that 5-fluorouracil (5-FU), a commonly used antimetabolite therapeutic with varying efficacy, induces specific alterations to nucleotide metabolism by disrupting pyrimidine homeostasis. An integrative metabolomics analysis of the cellular response to 5-FU reveals intracellular uracil accumulation, whereas deoxyuridine levels exhibited increased flux into the extracellular space, resulting in an induction of overflow metabolism. Subsequent analysis from mice bearing colorectal tumors treated with 5-FU show specific secretion of metabolites in tumor-bearing mice into serum that results from alterations in nucleotide flux and reduction in overflow metabolism. Together, these findings identify a determinant of an antimetabolite response that may be exploited to more precisely define the tumors that could respond to targeting cancer metabolism.

The successful extraction of metabolites is a critical step in metabolite profiling. By optimizing metabolite extraction, the range and quantitative capacity of metabolomics studies can be improved. We considered eight separate extraction protocols for the preparation of a metabolite extract from cultured mammalian cells. Parameters considered included temperature, pH, and cell washing before extraction. The effects on metabolite recovery were studied using a liquid chromatography high-resolution mass spectrometry (LC-HRMS) platform that measures metabolites of diverse chemical classes, including amino acids, lipids, and sugar derivatives. The temperature considered during the extraction or the presence of formic acid, a commonly used additive, was shown to have minimal effects on the measured ion intensities of metabolites. However, washing of samples before metabolite extraction, whether with water or phosphate-buffered saline, exhibited dramatic effects on measured intensities of both intracellular and extracellular metabolites. Together, these findings present a systematic assessment of extraction conditions for metabolite profiling.

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective. DOI: http://dx.doi.org/10.7554/eLife.03342.001, eLife digest Cells generate energy from a sugar called glucose via a process called glycolysis. This process involves many enzymes that catalyze 10 different chemical reactions, and it essentially converts glucose step-by-step into a simpler chemical called pyruvate. Pyruvate is then normally transported into structures within the cell called mitochondria, where it is further broken down using oxygen to release more energy. However, in cells that are rapidly dividing, pyruvate is converted into another chemical called lactate—which releases energy more quickly, but releases less energy overall. Cancer cells often convert most of their glucose into lactate, rather than breaking down pyruvate in their mitochondria: an observation known as the ‘Warburg effect’. And while many factors affect how a cell releases energy from pyruvate, it remains unclear what regulates which of these biochemical processes is most common in a living cell. In this study, Shestov et al. have developed a computational model for the process of glycolysis and used this to investigate the causes of the Warburg Effect. The model was based on the known characteristics of the enzymes and chemical reactions involved at each step. It predicted that the activity of the enzyme called GAPDH, which carries out the sixth step in glycolysis, in many cases affects how much lactate is produced. This suggests that this enzyme represents a bottleneck in the pathway. Next, Shestov et al. performed experiments where they used drugs to block different stages of the glycolysis pathway, and confirmed that the GAPDH enzyme is important for regulating this pathway in living cancer cells too. In these treated cells, the levels of a chemical called fructose-1,6-biphosphate (which is made in a step in the pathway between glucose and pyruvate) were either very high or very low. Shestov et al. proposed that the flow of chemicals through the glycolysis pathway is controlled by the GAPDH enzyme when the chemicals used by the enzymes upstream of GAPDH in the pathway (which includes fructose-1,6-biphosphate) are plentiful. However, if these chemicals are limited, other enzymes that are involved in earlier steps of the pathway regulate the process instead. The findings of Shestov et al. reveal that the regulation of glycolysis is more complex than previously thought, and is also very different when cells are undergoing the Warburg Effect. In the future, these findings might help to identify the types of cancer that could be effectively treated using drugs that target the glycolysis process, which are currently being tested in pre-clinical studies. DOI: http://dx.doi.org/10.7554/eLife.03342.002

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously. Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Recent advances in mass spectrometry have allowed for unprecedented characterization of human metabolism and its contribution to disease. Despite these advances, limitations in metabolomics technology remain. Here, we describe a metabolomics strategy that consolidates several recent improvements in mass spectrometry technology. The platform involves a high-resolution Orbitrap mass spectrometer coupled to faster scanning speeds, allowing for polarity switching and improved ion optics resulting in enhanced sensitivity. When coupled to HILIC chromatography, we are able to quantify over 339 metabolites from an extract of HCT8 cells with a linear range of over 4 orders of magnitude in a single chromatographic run. These metabolites include diverse chemical classes ranging from amino acids to polar lipids. In addition, we also detect over 3000 additional potential metabolites present in mammalian cells. We applied this platform to characterize the metabolome of eight colorectal cancer cell lines and observed both commonalities and heterogeneities across their metabolic profiles when cells are grown in identical conditions. Together these results demonstrate that simultaneous profiling and quantitation of the human metabolome is feasible.

Although it has long been reported that mutations in ribosome proteins (RP) are associated with increased cancer risk in humans, the molecular basis why which RP mutations do so remains unclear. Nevertheless, the prevailing view is that RP mutations, such as Rps19, are thought to alter transformation potential through general impairment of ribosome biogenesis or function. Importantly, recent observations are beginning to challenge this notion as too simplistic. We have determined that the RP, Rpl22, is not essential for ribosome biogenesis or global protein synthesis; however, its inactivation impairs the development of normal T lymphocytes and increases their transformation potential. Indeed, RPL22 is inactivated in human T acute lymphoblastic leukemia (T-ALL) and this is associated with reduced survival. Moreover, Rpl22-deficiency accelerates development of leukemia in a myristylated Akt2 transgenic (MyrAkt2 Tg) mouse model of T-ALL. To gain insight into how Rpl22 inactivation facilitates development of leukemia, we are performing unbiased transcriptomic and proteomic analysis on Rpl22+/+ and Rpl22-/- thymic lymphomas arising in the MyrAkt2 Tg model, and in an Rpl22-/- lymphoma reconstituted with Rpl22. Interestingly, relatively few changes in mRNA transcript read depth were observed; however, substantial differences in the proteome were observed. Pathway analysis revealed that the loss of Rpl22 altered the expression of proteins regulating RNA-processing, in particular RNA-splicing. Interestingly, interrogation of the transrciptome data for alternative splicing revealed that alterations in exon usage. The ability of Rpl22 to influence splicing appears to be conserved across species as alternative splicing was also observed in zebrafish embryos in which Rpl22 was knocked down using morpholino oligonucleotides. Consequently, we hypothesize that Rpl22 regulates biological events through its ability of binding to RNA targets, and controlling the expression of their protein products at least in part through altering mRNA splicing. How Rpl22 changes mRNA splicing pattern is currently under investigation. Disclosures No relevant conflicts of interest to declare.