Chaperone-mediated autophagy sustains haematopoietic stem-cell function.

The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.

Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification.

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC-MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC-MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot's Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Pipecolic Acid, a Putative Mediator of the Encephalopathy of Cerebral Malaria and the Experimental Model of Cerebral Malaria.

BACKGROUND: We explored a metabolic etiology of cerebral malaria (CM) coma. METHODS: Plasma metabolites were compared between Malawian children with CM and mild Plasmodium falciparum malaria. A candidate molecule was further studied in animal models of malaria. RESULTS: Clinically abnormal concentrations of pipecolic acid (PA) were present in CM plasma, and nearly normal in mild malaria samples. PA is renally cleared and the elevated PA blood levels were associated with renal insufficiency, which was present only in CM subjects. Prior studies demonstrate that PA has neuromodulatory effects and is generated by malaria parasites. PA brain levels in Plasmodium berghei ANKA-infected animals in the experimental cerebral malaria (ECM) model inversely correlated with normal behavior and correlated with blood-brain barrier (BBB) permeability. Mice infected with malaria species that do not induce neurological abnormalities or manifest BBB permeability had elevated plasma PA levels similar to ECM plasma at 7 days postinfection; however, they had low PA levels in the brain compared to ECM mice brains at 7 days postinfection. CONCLUSIONS: Our model suggests that malaria-generated PA induces coma in CM and in ECM. The role of BBB permeability and the mechanisms of PA neuromodulation in CM will require additional investigation.

Comparative Monomethylarginine Proteomics Suggests that Protein Arginine Methyltransferase 1 (PRMT1) is a Significant Contributor to Arginine Monomethylation in Toxoplasma gondii.

Arginine methylation is a common posttranslational modification found on nuclear and cytoplasmic proteins that has roles in transcriptional regulation, RNA metabolism and DNA repair. The protozoan parasite Toxoplasma gondii has a complex life cycle requiring transcriptional plasticity and has unique transcriptional regulatory pathways. Arginine methylation may play an important part in transcriptional regulation and splicing biology in this organism. The T. gondii genome contains five putative protein arginine methyltransferases (PRMTs), of which PRMT1 is important for cell division and growth. In order to better understand the function(s) of the posttranslational modification monomethyl arginine (MMA) in T. gondii, we performed a proteomic analysis of MMA proteins using affinity purification employing anti-MMA specific antibodies followed by mass spectrometry. The arginine monomethylome of T. gondii contains a large number of RNA binding proteins and multiple ApiAP2 transcription factors, suggesting a role for arginine methylation in RNA biology and transcriptional regulation. Surprisingly, 90% of proteins that are arginine monomethylated were detected as being phosphorylated in a previous phosphoproteomics study which raises the possibility of interplay between MMA and phosphorylation in this organism. Supporting this, a number of kinases are also arginine methylated. Because PRMT1 is thought to be a major PRMT in T. gondii, an organism which lacks a MMA-specific PRMT, we applied comparative proteomics to understand how PRMT1 might contribute to the MMA proteome in T. gondii We identified numerous putative PRMT1 substrates, which include RNA binding proteins, transcriptional regulators (e.g. AP2 transcription factors), and kinases. Together, these data highlight the importance of MMA and PRMT1 in arginine methylation in T. gondii, as a potential regulator of a large number of processes including RNA biology and transcription.

The Methods Employed in Mass Spectrometric Analysis of Posttranslational Modifications (PTMs) and Protein-Protein Interactions (PPIs).

Mass Spectrometry (MS) has revolutionized the way we study biomolecules, especially proteins, their interactions and posttranslational modifications (PTM). As such MS has established itself as the leading tool for the analysis of PTMs mainly because this approach is highly sensitive, amenable to high throughput and is capable of assigning PTMs to specific sites in the amino acid sequence of proteins and peptides. Along with the advances in MS methodology there have been improvements in biochemical, genetic and cell biological approaches to mapping the interactome which are discussed with consideration for both the practical and technical considerations of these techniques. The interactome of a species is generally understood to represent the sum of all potential protein-protein interactions. There are still a number of barriers to the elucidation of the human interactome or any other species as physical contact between protein pairs that occur by selective molecular docking in a particular spatiotemporal biological context are not easily captured and measured.PTMs massively increase the complexity of organismal proteomes and play a role in almost all aspects of cell biology, allowing for fine-tuning of protein structure, function and localization. There are an estimated 300 PTMS with a predicted 5% of the eukaryotic genome coding for enzymes involved in protein modification, however we have not yet been able to reliably map PTM proteomes due to limitations in sample preparation, analytical techniques, data analysis, and the substoichiometric and transient nature of some PTMs. Improvements in proteomic and mass spectrometry methods, as well as sample preparation, have been exploited in a large number of proteome-wide surveys of PTMs in many different organisms. Here we focus on previously published global PTM proteome studies in the Apicomplexan parasites T. gondii and P. falciparum which offer numerous insights into the abundance and function of each of the studied PTM in the Apicomplexa. Integration of these datasets provide a more complete picture of the relative importance of PTM and crosstalk between them and how together PTM globally change the cellular biology of the Apicomplexan protozoa. A multitude of techniques used to investigate PTMs, mostly techniques in MS-based proteomics, are discussed for their ability to uncover relevant biological function.

Buprenorphine decreases the CCL2-mediated chemotactic response of monocytes.

Despite successful combined antiretroviral therapy, ∼ 60% of HIV-infected people exhibit HIV-associated neurocognitive disorders (HAND). CCL2 is elevated in the CNS of infected people with HAND and mediates monocyte influx into the CNS, which is critical in neuroAIDS. Many HIV-infected opiate abusers have increased neuroinflammation that may augment HAND. Buprenorphine is used to treat opiate addiction. However, there are few studies that examine its impact on HIV neuropathogenesis. We show that buprenorphine reduces the chemotactic phenotype of monocytes. Buprenorphine decreases the formation of membrane projections in response to CCL2. It also decreases CCL2-induced chemotaxis and mediates a delay in reinsertion of the CCL2 receptor, CCR2, into the cell membrane after CCL2-mediated receptor internalization, suggesting a mechanism of action of buprenorphine. Signaling pathways in CCL2-induced migration include increased phosphorylation of p38 MAPK and of the junctional protein JAM-A. We show that buprenorphine decreases these phosphorylations in CCL2-treated monocytes. Using DAMGO, CTAP, and Nor-BNI, we demonstrate that the effect of buprenorphine on CCL2 signaling is opioid receptor mediated. To identify additional potential mechanisms by which buprenorphine inhibits CCL2-induced monocyte migration, we performed proteomic analyses to characterize additional proteins in monocytes whose phosphorylation after CCL2 treatment was inhibited by buprenorphine. Leukosialin and S100A9 were identified and had not been shown previously to be involved in monocyte migration. We propose that buprenorphine limits CCL2-mediated monocyte transmigration into the CNS, thereby reducing neuroinflammation characteristic of HAND. Our findings underscore the use of buprenorphine as a therapeutic for neuroinflammation as well as for addiction.

Identification of cell-specific targets of sumoylation during mouse spermatogenesis.

Recent findings suggest diverse and potentially multiple roles of small ubiquitin-like modifier (SUMO) in testicular function and spermatogenesis. However, SUMO targets remain uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from freshly purified spermatocytes and spermatids. One-hundred and twenty proteins were uniquely identified in the spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell-cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization, and in vitro sumoylation studies. GPS-SUMO Software was used to identify consensus and non-consensus sumoylation sites within the amino acid sequences of the proteins. The analyses confirmed the cell-specific sumoylation and/or SUMO interaction of several novel, previously uncharacterized SUMO targets such as CDK1, RNAP II, CDC5, MILI, DDX4, TDP-43, and STK31. Furthermore, several proteins that were previously identified as SUMO targets in somatic cells (KAP1 and MDC1) were identified as SUMO targets in germ cells. Many of these proteins have a unique role in spermatogenesis and during meiotic progression. This research opens a novel avenue for further studies of SUMO at the level of individual targets.

Can your protein be sumoylated? A quick summary and important tips to study SUMO-modified proteins.

A diverse set of SUMO target proteins has been identified. Therefore, there is a growing interest in studying sumoylation and SUMO interactions in cells. When the sumoylation of a protein or a SUMO interaction is suspected, a standard co-immunoprecipitation analysis using anti-SUMO and anti-target protein antibody is usually performed as a first step. However, the identification of endogenous sumoylated proteins is challenging because of the activity of isopeptidases, and often only a small fraction of a target protein is sumoylated at a given time. Here, we briefly summarize several important steps to ensure a successful co-immunoprecipitation analysis to detect possible sumoylation.

Receptor-type protein-tyrosine phosphatase ζ is a functional receptor for interleukin-34.

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.

Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesicles.

Texas-Red-asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles.

Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification.

Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide-metal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Na2 or if 25 mM EDTA-Na2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of α-casein and ß-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either α- or ß-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the α-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 µg of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore ≥20). Of these sites 79% were mono-, 20% di-, and ∼1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.

Phospholipids trigger Cryptococcus neoformans capsular enlargement during interactions with amoebae and macrophages.

A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.

Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa.

BACKGROUND: Sumoylation is a type of post-translational modification that is implicated in the regulation of numerous cellular events. However, its role in the function of human sperm has not yet been characterized. METHODS AND RESULTS: In this study, both immunofluorescence and electron microscopy revealed that small ubiquitin-like modifiers (SUMO) SUMO1 and SUMO2/3 were highly enriched in the neck area of human sperm that is associated with the redundant nuclear envelope and were also detectable in the flagella and some head regions. Similar localization patterns of SUMO were also observed in mouse and fly sperm. Nonmotile, two-tailed, curled tailed, misshapen, microcephalic (small head) and aciphalic (no head) sperm exhibited abnormally high levels of sumoylation in their neck and tail regions relative to normal sperm. Numerous sumoylated proteins, ranging from 20 to 260 kDa, were detected via western blotting and identified by mass spectrometry, and 55 SUMO targets that were present specifically in human sperm, and not in the control fraction, corresponded to flagella proteins, proteins involved in the maturation and differentiation of sperm, heat shock proteins and important glycolytic and mitochondrial enzymes. The targets that were identified included proteins with specific functions in germ cells and sperm, such as heat shock-related 70-kDa protein 2, outer dense fiber protein 3, A-kinase anchor proteins 3 and 4, L-lactate dehydrogenase C, sperm protein associated with the nucleus on the X chromosome B/F, valosin-containing protein, seminogelins, histone H4 and ubiquitin. Coimmunoprecipitation experiments confirmed the sumoylation of semenogelin and indicated that some sperm proteins are modified by sumoylation and ubiquitination simultaneously. CONCLUSIONS: Numerous proteins are modified by sumoylation in human sperm; excessive sumoylation is a marker of defective spermatozoa.

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer "sandwich" gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies.

Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model.

BACKGROUND: Three-dimensional (3D) cell culture has emerged as an alternative approach to 2D flat culture to model more accurately the phenotype of solid tissue in laboratories. Culturing cells in 3D more precisely recapitulates physiological conditions of tissues, as these cells reduce activities related to proliferation, focusing their energy consumption toward metabolism and homeostasis. RESULTS: Here, we demonstrate that 3D liver spheroids are a suitable system to model chromatin dynamics and response to epigenetics inhibitors. To delay necrotic tissue formation despite proliferation arrest, we utilize rotating bioreactors that apply active media diffusion and low shearing forces. We demonstrate that the proteome and the metabolome of our model resemble typical liver functions. We prove that spheroids respond to sodium butyrate (NaBut) treatment, an inhibitor of histone deacetylases (HDACi), by upregulating histone acetylation and transcriptional activation. As expected, NaBut treatment impaired specific cellular functions, including the energy metabolism. More importantly, we demonstrate that spheroids reestablish their original proteome and transcriptome, including pre-treatment levels of histone acetylation, metabolism, and protein expression once the standard culture condition is restored after treatment. Given the slow replication rate (> 40 days) of cells in 3D spheroids, our model enables to monitor the recovery of approximately the same cells that underwent treatment, demonstrating that NaBut does not have long-lasting effects on histone acetylation and gene expression. These results suggest that our model system can be used to quantify molecular memory on chromatin. CONCLUSION: Together, we established an innovative cell culture system that can be used to model anomalously decondensing chromatin in physiological cell growth and rule out epigenetics inheritance if cells recover the original phenotype after treatment. The transient epigenetics effects demonstrated here highlight the relevance of using a 3D culture model system that could be very useful in studies requiring long-term drug treatment conditions that would not be possible using a 2D cell monolayer system.

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry.

MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14 Å was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19 Å of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.

Mass Spectrometry to Study Chromatin Compaction.

Chromatin accessibility is a major regulator of gene expression. Histone writers/erasers have a critical role in chromatin compaction, as they "flag" chromatin regions by catalyzing/removing covalent post-translational modifications on histone proteins. Anomalous chromatin decondensation is a common phenomenon in cells experiencing aging and viral infection. Moreover, about 50% of cancers have mutations in enzymes regulating chromatin state. Numerous genomics methods have evolved to characterize chromatin state, but the analysis of (in)accessible chromatin from the protein perspective is not yet in the spotlight. We present an overview of the most used approaches to generate data on chromatin accessibility and then focus on emerging methods that utilize mass spectrometry to quantify the accessibility of histones and the rest of the chromatin bound proteome. Mass spectrometry is currently the method of choice to quantify entire proteomes in an unbiased large-scale manner; accessibility on chromatin of proteins and protein modifications adds an extra quantitative layer to proteomics dataset that assist more informed data-driven hypotheses in chromatin biology. We speculate that this emerging new set of methods will enhance predictive strength on which proteins and histone modifications are critical in gene regulation, and which proteins occupy different chromatin states in health and disease.

Mutational and functional analysis of Large in a novel CHO glycosylation mutant.

Inactivating mutations of Large reduce the functional glycosylation of alpha-dystroglycan (alpha-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT), and the C-terminal domain is related to the human i blood group transferase beta1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, alpha-DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the beta1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional alpha-DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional alpha-DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally glycosylated alpha-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of alpha-DG.

Nonprotein based enrichment method to analyze peptide cross-linking in protein complexes.

Cross-linking analysis of protein complexes and structures by tandem mass spectrometry (MS/MS) has advantages in speed, sensitivity, specificity, and the capability of handling complicated protein assemblies. However, detection and accurate assignment of the cross-linked peptides are often challenging due to their low abundance and complicated fragmentation behavior in collision-induced dissociation (CID). To simplify the MS analysis and improve the signal-to-noise ratio of the cross-linked peptides, we developed a novel peptide enrichment strategy that utilizes a cross-linker with a cryptic thiol group and using beads modified with a photocleavable cross-linker. The functional cross-linkers were designed to react with the primary amino groups in proteins. Human serum albumin was used as a model protein to detect intra- and intermolecular cross-linkages. Use of this protein-free selective retrieval method eliminates the contamination that can result from avidin-biotin based retrieval systems and simplifies data analysis. These features may make the method suitable to investigate protein-protein interactions in biological samples.