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Although the O-linked N-acetylglucosamine (O-GlcNAc) modification of the RNA polymerase II C-terminal domain was described 20 years ago, the function of this RNA polymerase II (pol II) species is not known. We show here that an O-GlcNAcylated pol II species (pol II) exists on promoters in vitro. Inhibition of O-GlcNAc-transferase activity and O-GlcNAcylation prevents pol II entry into the promoter, and O-GlcNAc removal from pol II is an ATP-dependent step during initiation. These data indicate thatO-GlcNAc-transferase activity is essential for RNA pol II promoter recruitment and that pol II goes through a cycling of O-GlcNAcylation at the promoter. Mass spectrometry shows that serine residues 2 and 5 of the pol II C-terminal domain are O-GlcNAcylated, suggesting an overlap with the transcription factor IIH (TFIIH)-dependent serine 5 phosphorylation events during initiation and P-TEFb (positive transcriptional elongation factor b) events during elongation. These data provide unexpected and important insights into the role of a previously ill-defined species of RNA polymerase II in regulating transcription. [ABSTRACT FROM AUTHOR]

Stored waveform inverse Fourier transform (SWIFT) technology has been implemented on a commercial Fourier transform ICR mass spectrometer. Complex ejection/isolation waveforms can be generated on an arbitrary waveform generator (AWG) and applied to the instrument by use of a high speed analog switch. High mass selectivity and subsequent electron capture dissociation (ECD) of the SWIFT isolated ions has been demonstrated with analysis of intact Bovine histone H4. A mass selectivity about 0.1 m/z unit for isolation of the 18+ charge state ions was achieved. Adaptation of SWIFT on the commercial instrument provides an enhanced capability for characterizing intact proteins by top-down analysis. [Copyright &y& Elsevier]

During the second half of the 20th century, biochemistry and subsequently molecular biology blossomed into the core upon which all biological and biomedical sciences now depend. A major part of these closely related disciplines has been the study of the structure and function of proteins and the diverse biological functions that they perform. Early experimentation necessarily focused on individual entities, selected mainly for their activities, but as technology improved there developed a tendency to look at proteins as larger, interactive groups or clusters. Spurred by the recent exponential production of genomic sequence data for a rapidly increasing number of species, protein chemistry has now evolved into a new discipline, proteomics. In addition to embracing the methods and approaches that have served protein scientists well in the past, it includes, and is perhaps best defined by, high-throughput analyses based in large part on 2D gel electrophoresis, MALDI and ESI mass spectrometry and combinatorial arrays. Proteomic targets include the identification of all genome products and a mapping of their interactions and expression profiles. These hold great promise for the identification of disease markers and drug targets, but are not without their challenges and pitfalls. IUBMB Life, 57: 267–272, 2005 [ABSTRACT FROM AUTHOR]

[1-²H2]Ethanol, [2-²H3]ethanol and [1-13C, 1-²H2]ethanol were administered to bile fistula rats under conditions ensuring a maximal rate of oxidation for about 24 h. Cholesterol, cholic acid, chenodeoxycholic acid and 3β,11β,21-trihydroxy-5α-pregnan-20-one were isolated from bile collected at intervals during the experiment. The fraction of molecules containing different numbers of heavy isotope atoms was measured by gas chromatography-mass spectrometry-computer techniques. The results were used to determine the origin of the cholesterol pools used as precursors for the different compounds isolated from bile. The bile acids were found to be formed from a cholesterol pool, the turnover of which was about 5 times more rapid than that of the cholesterol pool used for biliary excretion. The cholesterol pools used for biliary excretion and as corticosterone precursor seemed to be in equilibrium. The acetyl groups derived from ethanol were introduced into the steroids without exchange of hydrogens at C-2, and the acetyl-CoA pool used as precursor did not exchange with acetyl groups in e.g. citrate or fatty acids. A difference in incorporation of [2-²H3]acetyl groups and [1-13C]acetyl groups was interpreted to be due to an isotope effect. Ethanol contributed about 45% of the acetvl-CoA pool used as precursor of cholesterol and bile acids. Deuterium from [1-²H2]ethanol was incorporated in many positions of all the steroids. If it is assumed that the incorporation is mediated by NADPH, the deuterium excess of the hydrogens introduced was about 10 atoms %. The contribution via different pathways of an important part of the hydrogen and carbon atoms in cholesterol, bile acids and corticosteroids provides possible mechanisms by which ethanol may affect the biosynthesis and metabolism of steroids. [ABSTRACT FROM AUTHOR]

Vertebrate nervous systems use the axon initial segment (AIS) to initiate action potentials and maintain neuronal polarity. The microtubule-associated protein tripartite motif containing 46 (TRIM46) was reported to regulate axon specification, AIS assembly, and neuronal polarity through the bundling, or fasciculation, of microtubules in the proximal axon. However, these claims are based on TRIM46 knockdown in cultured neurons. To investigate TRIM46 function in vivo, we examined male and female TRIM46 knock-out mice. Contrary to previous reports, we find that TRIM46 is dispensable for axon specification and AIS formation. TRIM46 knock-out mice are viable, have normal behavior, and have normal brain structure. Thus, TRIM46 is not required for AIS formation, axon specification, or nervous system function. However, we confirm that TRIM46 is required for microtubule fasciculation. We also show TRIM46 enrichment in the first ~100 µm of axon occurs independently of ankyrinG (AnkG) in vivo, although AnkG is required to restrict TRIM46 only to the AIS. Our results highlight the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function. [ABSTRACT FROM AUTHOR]

In mammals, interactions between the bone marrow (BM) stroma and hematopoietic progenitors contribute to bone-BM homeostasis. Perinatal bone growth and ossification provide a microenvironment for the transition to definitive hematopoiesis; however, mechanisms and interactions orchestrating the development of skeletal and hematopoietic systems remain largely unknown. Here, we establish intracellular O-linked ß-N-acetylglucosamine (O-GlcNAc) modification as a posttranslational switch that dictates the differentiation fate and niche function of early BM stromal cells (BMSCs). By modifying and activating RUNX2, O-GlcNAcylation promotes osteogenic differentiation of BMSCs and stromal IL-7 expression to support lymphopoiesis. In contrast, C/EBPß-dependent marrow adipogenesis and expression of myelopoietic stem cell factor (SCF) is inhibited by O-Glc-NAcylation. Ablating O-GlcNAc transferase (OGT) in BMSCs leads to impaired bone formation, increased marrow adiposity, as well as defective B-cell lymphopoiesis and myeloid overproduction in mice. Thus, the balance of osteogenic and adipogenic differentiation of BMSCs is determined by reciprocal O-GlcNAc regulation of transcription factors, which simultaneously shapes the hematopoietic niche. [ABSTRACT FROM AUTHOR]

Elucidating enzyme–substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme–substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein–protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation. Combining TurboID-mediated proximity labeling with quantitative phosphoproteomics identifies BIN2 signaling components including kinase substrates in vivo, revealing cellular functions of BIN2. [ABSTRACT FROM AUTHOR]

TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-?B master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPaseactivating protein (GAP) enzymes, and is exclusive to membranelocalized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection. [ABSTRACT FROM AUTHOR]

Protein–protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ( 1 O 2 ) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin–thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein–protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling. [ABSTRACT FROM AUTHOR]

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including Dhx30 and Llph, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed that RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts. [Display omitted] • Tag-free method to enrich ribosomes and associated proteins from any sample • Dhx30 and Elavl2 are embryonic forebrain ribosome-associated proteins (RAPs) • The RAP Llph may be important for neural development and translation of long mRNAs • Macrophages exhibit ribosome composition changes upon different stimuli Susanto et al. developed a tag-free method to characterize ribosome-associated proteins from any sample. They identified hundreds of candidate ribosome-associated proteins, some of which were dynamic across embryonic tissues or during responses to cell stimuli. These included proteins that may be important for neural development or during immune activation. [ABSTRACT FROM AUTHOR]

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. [ABSTRACT FROM AUTHOR]

Summary: The brassinosteroid (BR) signaling pathway includes two receptor-like kinases (BRI1 and BAK1), a plasma membrane-associated kinase (BSK1), two phosphatases (BSU1 and PP2A), a GSK3-like kinase (BIN2), and two homologous transcription factors (BZR1 and BES1/BZR2). But the mechanisms of signal relay are not fully understood. Here, we show that a receptor-like cytoplasmic kinase named CDG1 mediates signal transduction from BRI1 to BSU1. Transgenic experiments confirm that CDG1 and its homolog CDL1 positively regulate BR signaling and plant growth. Mass spectrometry analysis identified BRI1 phosphorylation sites in CDG1 and CDG1 phosphorylation sites in BSU1. Mutations of these phosphorylation sites compromised the BR signaling functions. The results demonstrate that BRI1 phosphorylates S234 to activate CDG1 kinase, and CDG1 in turn phosphorylates S764 to activate BSU1, which inactivates BIN2 by dephosphorylating Y200 of BIN2. This study thus demonstrates a complete phosphorylation/dephosphorylation cascade linking a steroid-activated receptor kinase to a GSK3-like kinase in plants. [ABSTRACT FROM AUTHOR]

The monosaccharide addition of an N-acetylglucosamine to serine and threonine residues of nuclear and cytosolic proteins (O-GlcNAc) is a posttranslational modification emerging as a general regulator of many cellular processes, including signal transduction, cell division, and transcription. The sole mouse O-GlcNAc transferase (OGT) is essential for embryonic development. To understand the role of OGT in mouse development better, we mapped sites of O-GlcNAcylation of nuclear proteins in mouse embryonic stem cells (ESCs). Here, we unambiguously identify over 60 nuclear proteins as O-GlcNAcylated, several of which are crucial for mouse ESC cell maintenance. Furthermore, we extend the connection between OGT and Polycomb group genes from flies to mammals, showing Polycomb repressive complex 2 is necessary to maintain normal levels of OGT and for the correct cellular distribution of O-GlcNAc. Together, these results provide insight into how OGT may regulate transcription in early development, possibly by modifying proteins important to maintain the ESC transcriptional repertoire. [ABSTRACT FROM AUTHOR]

Mediator is a multisubunit coactivator complex that facilitates transcription of nuclear receptors. We investigated the role of the mediator complex as a coactivator for vitamin D receptor (VDR) in keratinocytes. Using VDR affinity beads, the vitamin D receptor interacting protein (DRIP)/mediator complex was purified from primary keratinocytes, and its subunit composition was determined by mass spectrometry. The complex included core subunits, such as DRIP205/MED1 (MED1), that directly binds to VDR. Additional subunits were identified that are components of the RNA polymerase II complex. The functions of different mediator components were investigated by silencing its subunits. The core subunit MED1 facilitates VDR activity and regulating keratinocyte proliferation and differentiation. A newly described subunit MED21 also has a role in promoting keratinocyte proliferation and differentiation, whereas MED10 has an inhibitory role. Blocking MED1/MED21 expression caused hyperproliferation of keratinocytes, accompanied by increases in mRNA expression of the cell cycle regulator cyclin D1 and/or glioma-associated oncogene homolog. Blocking MED1 or MED21 expression also resulted in defects in calcium-induced keratinocyte differentiation, as indicated by decreased expression of differentiation markers and decreased translocation of E-cadherin to the membrane. These results show that keratinocytes use the transcriptional coactivator mediator to regulate VDR functions and control keratinocyte proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Densin-180 is a core component of post-synaptic densities, the highly complex molecular assemblies that mediate signaling between neuronal cells. It is a multi-domain scaffold protein characterized by multiple leucine-rich repeat domains plus a single Psd95/Discs large/Zona occludens-1 domain. In its original topology model a single transmembrane segment was proposed with an extracellular N-terminus and an intracellular C-terminus. However, recently discovered in vivo phosphorylation sites are incompatible with this topology. Here, we discuss an all-intracellular and membrane-associated localization of Densin-180 that is consistent with and supported by all the latest experimental data. This revised topology which now includes also a phosphorylation-rich area will have deciding influence on future research involving Densin-180 and its signaling. [ABSTRACT FROM AUTHOR]

Ferroptosis is a caspase-independent, iron-dependent form of regulated necrosis extant in traumatic brain injury, Huntington disease, and hemorrhagic stroke. It can be activated by cystine deprivation leading to glutathione depletion, the insufficiency of the antioxidant glutathione peroxidase-4, and the hemolysis products hemoglobin and hemin. A cardinal feature of ferroptosis is extracellular signal-regulated kinase (ERK)1/2 activation culminating in its translocation to the nucleus. We have previously confirmed that the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor U0126 inhibits persistent ERK1/2 phosphorylation and ferroptosis. Here, we show that hemin exposure, a model of secondary injury in brain hemorrhage and ferroptosis, activated ERK1/2 in mouse neurons. Accordingly, MEK inhibitor U0126 protected against hemin-induced ferroptosis. Unexpectedly, U0126 prevented hemin-induced ferroptosis independent of its ability to inhibit ERK1/2 signaling. In contrast to classical ferroptosis in neurons or cancer cells, chemically diverse inhibitors of MEK did not block hemin-induced ferroptosis, nor did the forced expression of the ERK-selective MAP kinase phosphatase (MKP)3. We conclude that hemin or hemoglobin-induced ferroptosis, unlike glutathione depletion, is ERK1/2-independent. Together with recent studies, our findings suggest the existence of a novel subtype of neuronal ferroptosis relevant to bleeding in the brain that is 5-lipoxygenase-dependent, ERK-independent, and transcription-independent. Remarkably, our unbiased phosphoproteome analysis revealed dramatic differences in phosphorylation induced by two ferroptosis subtypes. As U0126 also reduced cell death and improved functional recovery after hemorrhagic stroke in male mice, our analysis also provides a template on which to build a search for U0126's effects in a variant of neuronal ferroptosis. [ABSTRACT FROM AUTHOR]

Hirsch, Jan, Kirk C. Hansen, Alma L. Burlingame, and Michael A. Matthay. Proteomics: current techniques and potential applications to lung disease. Am J Physiol Lung Cell Mol Physiol 287: L1-L23, 2004; 10.1152/ajplung. 00301.2003.--Proteomics aims to study the whole protein content of a biological sample in one set of experiments. Such an approach has the potential value to acquire an understanding of the complex responses of an organism to a stimulus. The large vascular and air space surface area of the lung expose it to a multitude of stimuli that can trigger a variety of responses by many different cell types. This complexity makes the lung a promising, but also challenging, target for proteomics. Important steps made in the last decade have increased the potential value of the results of proteomics studies for the clinical scientist. Advances in protein separation and staining techniques have improved protein identification to include the least abundant proteins. The evolution in mass spectrometry has led to the identification of a large part of the proteins of interest rather than just describing changes in patterns of protein spots. Protein profiling techniques allow the rapid comparison of complex samples and the direct investigation of tissue specimens. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. These methodologies have made the application of proteomics on the study of specific diseases or biological processes under clinically relevant conditions possible. The quantity of data that is acquired with these new techniques places new challenges on data processing and analysis. This article provides a brief review of the most promising proteomics methods and some of their applications to pulmonary research. [ABSTRACT FROM AUTHOR]

The article focuses on TSC1 loss increases risk for tauopathy by inducing tau acetylation and preventing tau clearance via chaperone-mediated autophagy. Topics include the age-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions are known as tauopathies, the linked a loss-of-function mutation in the TSC1 gene to tau accumulation lobar degeneration, and the genetic variants in TSC1 that decrease TSC1/hamartin levels and predispose to tauopathies.

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition. [ABSTRACT FROM AUTHOR]

Presents a study which examined protein interactions by oxidative cross-linking of a glycine-glycine-histidine (GGH) fusion protein. Demonstration that GGH fused to the amino terminus of a protein can still support cross-linking; Factors which contribute to the cross-linking reaction; Method used in this study; Results of this study.

Neuronal ankyrins cluster and link membrane proteins to the actin and spectrin-based cytoskeleton. Among the three vertebrate ankyrins, little is known about neuronal Ankyrin-R (AnkR). We report AnkR is highly enriched in Pv+ fast-spiking interneurons in mouse and human. We identify AnkR-associated protein complexes including cytoskeletal proteins, cell adhesion molecules (CAMs), and perineuronal nets (PNNs). We show that loss of AnkR from forebrain interneurons reduces and disrupts PNNs, decreases anxiety-like behaviors, and changes the intrinsic excitability and firing properties of Pv+ fast-spiking interneurons. These changes are accompanied by a dramatic reduction in Kv3.1b K+ channels. We identify a novel AnkR-binding motif in Kv3.1b, and show that AnkR is both necessary and sufficient for Kv3.1b membrane localization in interneurons and at nodes of Ranvier. Thus, AnkR regulates Pv+ fast-spiking interneuron function by organizing ion channels, CAMs, and PNNs, and linking these to the underlying β1 spectrin-based cytoskeleton. [ABSTRACT FROM AUTHOR]

Background: Infants born at extremely low gestational age are at high risk for bronchopulmonary dysplasia and continuing lung disease. There are no early clinical biomarkers for pulmonary outcome and limited therapeutic interventions. Objectives: We performed global proteomics of premature infant tracheal aspirate (TA) and plasma to determine the composition and source of lung fluid proteins and to identify potential biomarkers of respiratory outcome. Methods: TA samples were collected from intubated infants in the TOLSURF cohort before and after nitric oxide treatment, and plasma was collected from NO CLD infants. Protein abundance was assayed by HPLC/tandem mass spectrometry and Protein Prospector software. mRNA abundance in mid-gestation fetal lung was assessed by RNA sequencing. Pulmonary morbidity was defined as a need for ventilatory support at term and during the first year. Results: Abundant TA proteins included albumin, hemoglobin, and actin-related proteins. 96 of 137 detected plasma proteins were present in TA (r = 0.69, p<0.00001). Based on lung RNAseq data, ~88% of detected TA proteins in injured infant lung are derived at least in part from lung epithelium with overrepresentation in categories of cell membrane/secretion and stress/inflammation. Comparing 37 infants at study enrollment (7–14 days) who did or did not develop persistent pulmonary morbidity, candidate biomarkers of both lung (eg., annexin A5) and plasma (eg., vitamin D-binding protein) origin were identified. Notably, levels of free hemoglobin were 2.9-fold (p = 0.03) higher in infants with pulmonary morbidity. In time course studies, hemoglobin decreased markedly in most infants after enrollment coincident with initiation of inhaled nitric oxide treatment. Conclusions: We conclude that both lung epithelium and plasma contribute to the lung fluid proteome in premature infants with lung injury. Early postnatal elevation of free hemoglobin and heme, which are both pro-oxidants, may contribute to persistent lung disease by depleting nitric oxide and increasing oxidative/nitrative stress. [ABSTRACT FROM AUTHOR]

NGLY1 is a widely conserved eukaryotic cytosolic deglycosylating enzyme involved in the endoplasmic reticulum-associated degradation (ERAD) process, which eliminates misfolded proteins through retrograde translocation and proteasomal degradation. A human genetic disorder called NGLY1-deficiency has been reported, indicating the functional importance of NGLY1 in humans. Evidence suggests that Ngly1 -KO is embryonic lethal in mice, while additional deletion of the Engase gene, encoding another cytosolic deglycosylating enzyme (endo-β- N -acetylglucosaminidase; ENGase), partially rescued lethality. Upon compromised Ngly1 activity, ENGase-mediated deglycosylation of misfolded glycoproteins may cause excess formation of N -GlcNAc proteins in the cytosol, leading to detrimental effects in the mice. Whether endogenous N -GlcNAc proteins are really formed in Ngly1 -KO cells/animals or not remains unclarified. Here, comprehensive identification of O - and N- GlcNAc proteins was carried out using purified cytosol from wild type, Ngly1 -KO, Engase -KO, and Ngly1/Engase double KO mouse embryonic fibroblasts. It was revealed that while there is no dramatic change in the level of O -GlcNAc proteins among cells examined, there was a vast increase of N- GlcNAc proteins in Ngly1 -KO cells upon proteasome inhibition. Importantly, few N- GlcNAc proteins were observed in Engase -KO or Ngly1/Engase double-KO cells, clearly indicating that the cytosolic ENGase is responsible for the formation of N- GlcNAc proteins. The excess formation of N- GlcNAc proteins may at least in part account for the pathogenesis of NGLY1-deficiency. Image 1 • In Ngly1 -KO, ENGase is responsible for the formation of cytosolic N -GlcNAc proteins. • Knockout of both Ngly1 and Engase decreases the level of cytosolic N- GlcNAc proteins. • Identified 3000 unique O- GlcNAcylated peptides and 695 unambiguous O -GlcNAc sites. [ABSTRACT FROM AUTHOR]

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis. [ABSTRACT FROM AUTHOR]

Increased neural stem cell (NSC) quiescence is a major determinant of age-related regenerative decline in the adult hippocampus. However, a coextensive model has been proposed in which divisioncoupled conversion of NSCs into differentiated astrocytes restrict the stem cell pool with age. Here we report that age-related loss of the posttranslational modification, O-linked ß-N-acetylglucosamine (O-GlcNAc), in NSCs promotes a glial fate switch. We detect an age-dependent decrease in NSC O-GlcNAc levels coincident with decreased neurogenesis and increased gliogenesis in themature hippocampus. Mimicking an age-related loss of NSC O-GlcNAcylation in young mice reduces neurogenesis, increases astrocyte differentiation, and impairs associated cognitive function. Using RNAsequencing of primary NSCs following decreased O-GlcNAcylation, we detected changes in the STAT3 signaling pathway indicative of glial differentiation. Moreover, using O-GlcNAc-specific mass spectrometry analysis of the aging hippocampus, together with an in vitro site-directedmutagenesis approach, we identify loss of STAT3 O-GlcNAc at Threonine 717 as a driver of astrocyte differentiation. Our data identify the posttranslational modification, O-GlcNAc, as a key molecular regulator of regenerative decline underlying an age-related NSC fate switch. [ABSTRACT FROM AUTHOR]

Axon initial segments (AISs) initiate action potentials and regulate the trafficking of vesicles between somatodendritic and axonal compartments. However, the mechanisms controlling AIS assembly remain poorly defined. We performed differential proteomics and found nuclear mitotic apparatus protein 1 (NuMA1) is downregulated in AIS-deficient neonatal mouse brains and neurons. NuMA1 is transiently located at the AIS during development where it interacts with the scaffolding protein 4.1B and the dynein regulator lissencephaly 1 (Lis1). Silencing NuMA1 or protein 4.1B by shRNA disrupts AIS assembly, but not maintenance. Silencing Lis1 or overexpressing NuMA1 during AIS assembly increased the density of AIS proteins, including ankyrinG and neurofascin-186 (NF186). NuMA1 inhibits the endocytosis of AIS NF186 by impeding Lis1's interaction with doublecortin, a potent facilitator of NF186 endocytosis. Our results indicate the transient expression and AIS localization of NuMA1 stabilizes the developing AIS by inhibiting endocytosis and removal of AIS proteins. [ABSTRACT FROM AUTHOR]

Despite the high and preferential expression of p38γ MAPK in the myocardium, little is known about its function in the heart. The aim of the current study was to elucidate the physiologic and biochemical roles of p38γ in the heart. Expression and subcellular localization of p38 isoforms was determined in mouse hearts. Comparisons of the cardiac function and structure of wild-type and p38γ knockout (KO) mice at baseline and after abdominal aortic banding demonstrated that KO mice developed less ventricular hypertrophy and that contractile function is better preserved. To identify potential substrates of p38γ, we generated an analog-sensitive mutant to affinity tag endogenous myocardial proteins. Among other proteins, this technique identified calpastatin as a direct p38γ substrate. Moreover, phosphorylation of calpastatin by p38γ impaired its ability to inhibit the protease, calpain. We have identified p38γ as an important determinant of the progression of pathologic cardiac hypertrophy after aortic banding in mice. In addition, we have identified calpastatin, among other substrates, as a novel direct target of p38γ that may contribute to the protection observed in p38γKO mice. [ABSTRACT FROM AUTHOR]

The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'κ constrains SIT1 activity under salt stress. B'κ-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'κ overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance. B'κ functions in a SIT1 kinase-dependent manner. During early salt stress, activated SIT1 phosphorylates B'κ; this not only enhances its binding with SIT1, it also promotes B'κ protein accumulation via Ser502 phosphorylation. Consequently, by blocking SIT1 phosphorylation, B'κ inhibits and fine-tunes SIT1 activity to balance plant growth and stress adaptation. [ABSTRACT FROM AUTHOR]

Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation. [ABSTRACT FROM AUTHOR]

Schwann cells (SCs), the glia of the peripheral nervous system, play an essential role in nerve regeneration. Upon nerve injury, SCs are reprogrammed into unique "repair SCs," and these cells remove degenerating axons/myelin debris, promote axonal regrowth, and ultimately remyelinate regenerating axons. The AP-1 transcription factor JUN is promptly induced in SCs upon nerve injury and potently mediates this injury-induced SC plasticity; however, the regulation of these JUN-dependent SC injury responses is unclear. Previously, we produced mice with a SC-specific deletion of OGlcNAc transferase (OGT). This enzyme catalyzes O-GlcNAcylation, a posttranslational modification that is influenced by the cellular metabolic state. Mice lacking OGT in SCs develop a progressive demyelinating peripheral neuropathy. Here, we investigated the nerve repair process in OGT-SCKO mutant mice and found that the remyelination of regenerating axons is severely impaired. Gene expression profiling of OGT-SCKO SCs revealed that the JUNdependent SC injury program was elevated in the absence of injury and failed to shut down at the appropriate time after injury. This aberrant JUN activity results in abnormalities in repair SC function and redifferentiation and prevents the timely remyelination. This aberrant nerve injury response is normalized in OGT-SCKO mice with reduced Jun gene dosage in SCs. Mechanistically, OGT O-GlcNAcylates JUN at multiple sites, which then leads to an attenuation of AP-1 transcriptional activity. Together, these results highlight the metabolic oversight of the nerve injury response via the regulation of JUN activity by O-GlcNAcylation, a pathway that could be important in the neuropathy associated with diabetes and aging. [ABSTRACT FROM AUTHOR]

How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size – regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin b 1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3 ′ untranslated region (3 ′ UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3 ′ UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling. [ABSTRACT FROM AUTHOR]

Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargylpuromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotinazide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography- tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment. [ABSTRACT FROM AUTHOR]

The N -methyl- d -aspartate-type glutamate receptor (NMDAR)-associated multiprotein complexes are indispensable for synaptic plasticity and cognitive functions. While purification and proteomic analyses of these signaling complexes have been performed in adult rodent and human brain, much less is known about the protein composition of NMDAR complexes in the developing brain and their modifications by neonatal hypoxic-ischemic (HI) brain injury. In this study, the postsynaptic density proteins were prepared from postnatal day 9 naïve, sham-operated and HI-injured mouse cortex. The GluN2B-containing NMDAR complexes were purified by immunoprecipitation with a mouse GluN2B antibody and subjected to mass spectrometry analysis for determination of the GluN2B binding partners. A total of 71 proteins of different functional categories were identified from the naïve animals as native GluN2B-interacting partners in the developing mouse brain. Neonatal HI reshaped the postsynaptic GluN2B interactome by recruiting new proteins, including multiple kinases, into the complexes; and modifying the existing associations within 1 h of reperfusion. The early responses of postsynaptic NMDAR complexes and their related signaling networks may contribute to molecular processes leading to cell survival or death, brain damage and/or neurological disorders in term infants with neonatal encephalopathy. [ABSTRACT FROM AUTHOR]

Spectrins form a submembranous cytoskeleton proposed to confer strength and flexibility to neurons and to participate in ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Neuronal spectrin cytoskeletons consist of diverse β subunits and αII spectrin. Although αII spectrin is found in neurons in both axonal and somatodendritic domains, using proteomics, biochemistry, and superresolution microscopy, we show that αII and (31V spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptanl f/f mice for deletion of CNS αII spectrin. We analyzed αII spectrin-deficient mice of both sexes and found that loss of all spectrin causes profound reductions in all β spectrins. αII spectrin-deficient mice die before 1 month of age and have disrupted AIS and many other neurological impairments including seizures, disrupted cortical lamination, and widespread neurodegeneration. These results demonstrate the importance of the spectrin cytoskeleton both at the AIS and throughout the nervous system. [ABSTRACT FROM AUTHOR]

Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana. Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level. [ABSTRACT FROM AUTHOR]

Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications. [ABSTRACT FROM AUTHOR]

Summary Abnormalities in dendritic spines are manifestations of several neurodevelopmental and psychiatric diseases. TAOK2 is one of the genes in the 16p11.2 locus, copy number variations of which are associated with autism and schizophrenia. Here, we show that the kinase activity of the serine/threonine kinase encoded by TAOK2 is required for spine maturation. TAOK2 depletion results in unstable dendritic protrusions, mislocalized shaft-synapses, and loss of compartmentalization of NMDA receptor-mediated calcium influx. Using chemical-genetics and mass spectrometry, we identified several TAOK2 phosphorylation targets. We show that TAOK2 directly phosphorylates the cytoskeletal GTPase Septin7, at an evolutionary conserved residue. This phosphorylation induces translocation of Septin7 to the spine, where it associates with and stabilizes the scaffolding protein PSD95, promoting dendritic spine maturation. This study provides a mechanistic basis for postsynaptic stability and compartmentalization via TAOK2-Sept7 signaling, with implications toward understanding the potential role of TAOK2 in neurological deficits associated with the 16p11.2 region. [ABSTRACT FROM AUTHOR]

Schwann cells (SCs), ensheathing glia of the peripheral nervous system, support axonal survival and function. Abnormalities in SC metabolism affect their ability to provide this support and maintain axon integrity. To further interrogate this metabolic influence on axon-glial interactions, we generated OGT-SCKO mice with SC-specific deletion of the metabolic/nutrient sensing protein O-GlcNAc transferase that mediates the O-linked addition ofN-acetylglucosamine (GlcNAc) moieties to Ser and Thr residues. The OGT-SCKO mice develop tomaculous demyelinating neuropathy characterized by focal thickenings of the myelin sheath (tomacula), progressive demyelination, axonal loss, and motor and sensory nerve dysfunction. Proteomic analysis identified more than 100 O-GlcNAcylated proteins in rat sciatic nerve, including Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans. PRX lacking O-GlcNAcylation is mislocalized within the myelin sheath of these mutant animals. Furthermore, phenotypes of OGT-SCKO and Prxdeficient mice are very similar, suggesting that metabolic control of PRX O-GlcNAcylation is crucial for myelin maintenance and axonal integrity. [ABSTRACT FROM AUTHOR]

Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3. [ABSTRACT FROM AUTHOR]

Summary Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1 G93A , activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1 G93A mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH -tTA/ tetO -hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. Video Abstract [ABSTRACT FROM AUTHOR]

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 OGlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC selfrenewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor. [ABSTRACT FROM AUTHOR]

Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases (RLCKs); however, the molecular mechanisms that create an effective on-off switch are unknown. The receptor kinase BR INSENSITIVE1 (BRI1) transduces brassinosteroid (BR) signal by phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs, which contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain. Here, we show that the BR signaling function of BSKs is conserved in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and that the TPR domain of BSKs functions as a "phospho-switchable" autoregulatory domain to control BSKs' activity. Genetic studies revealed that OsBSK3 is a positive regulator of BR signaling in rice, while in vivo and in vitro assays demonstrated that OsBRI1 interacts directly with and phosphorylates OsBSK3. The TPR domain of OsBSK3, which interacts directly with the protein's kinase domain, serves as an autoinhibitory domain to prevent OsBSK3 from interacting with bri1-SUPPRESSOR1 (BSU1). Phosphorylation of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains, thereby increasing the binding between OsBSK3's kinase domain and BSU1. Our results not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in rice, but also provide insight into the molecular mechanism by which BSK family proteins are inhibited under basal conditions but switched on by the upstream receptor kinase BRI1. [ABSTRACT FROM AUTHOR]

Brassinosteroid (BR) binding activates the receptor kinase BRI1 by inducing heterodimerization with its co-receptor kinase BAK1; however, the mechanisms that reversibly inactivate BRI1 remain unclear. Here we show that cytoplasm-localized protein phosphatase 2A (PP2A) B′ regulatory subunits interact with BRI1 to mediate its dephosphorylation and inactivation. Loss-of-function and overexpression experiments showed that a group of PP2A B′ regulatory subunits, represented by B′η, negatively regulate BR signaling by decreasing BRI1 phosphorylation. BR increases the expression levels of these B′ subunits, and B′η interacts preferentially with phosphorylated BRI1, suggesting that the dynamics of BR signaling are modulated by the PP2A-mediated feedback inactivation of BRI1. Compared with PP2A B′α and B′β, which promote BR responses by dephosphorylating the downstream transcription factor BZR1, the BRI1-inactivating B′ subunits showed similar binding to BRI1 and BZR1 but distinct subcellular localization. Alteration of the nuclear/cytoplasmic localization of the B′ subunits revealed that cytoplasmic PP2A dephosphorylates BRI1 and inhibits the BR response, whereas nuclear PP2A dephosphorylates BZR1 and activates the BR response. Our findings not only identify the PP2A regulatory B subunits that mediate the binding and dephosphorylation of BRI1, but also demonstrate that the subcellular localization of PP2A specifies its substrate selection and distinct effects on BR signaling. [ABSTRACT FROM AUTHOR]

The article discusses a study which examined the effect of the oligomerization between BSU1 family members on brassinosteroid signaling in Arabidopsis.

Cell surface receptors are central to the cell’s ability to generate coordinated responses to the multitude of biochemical and physical cues in the microenvironment. However, the mechanisms by which receptors enable this concerted cellular response remain unclear. To investigate the effect of cellular tension on cell surface receptors, we combined novel high- resolution imaging and single particle tracking with established biochemical assays to examine TGFβ signaling. We find that TGFβ receptors are discretely organized to segregated spatial domains at the cell surface. Integrin-rich focal adhesions organize TβRII around TβRI, limiting the integration of TβRII while sequestering TβRI at these sites. Disruption of cellular tension leads to a collapse of this spatial organization and drives formation of heteromeric TβRI/TβRII complexes and Smad activation. This work details a novel mechanism by which cellular tension regulates TGFβ receptor organization, multimerization, and function, providing new insight into the mechanisms that integrate biochemical and physical cues. [ABSTRACT FROM AUTHOR]

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function. [ABSTRACT FROM AUTHOR]

The microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Reducing tau levels ameliorates AD-related synaptic, network, and behavioral abnormalities in transgenic mice expressing human amyloid precursor protein (hAPP). We used mass spectrometry to characterize the post-translational modification of endogenous tau isolated from wild-type and hAPP mice. We identified seven types of tau modifications at 63 sites in wild-type mice. Wild-type and hAPP mice had similar modifications, supporting the hypothesis that neuronal dysfunction in hAPP mice is enabled by physiological forms of tau. Our findings provide clear evidence for acetylation and ubiquitination of the same lysine residues; some sites were also targeted by lysine methylation. Our findings refute the hypothesis of extensive O-linked N-acetylglucosamine (O-GlcNAc) modification of endogenous tau. The complex post-translational modification of physiological tau suggests that tau is regulated by diverse mechanisms. [ABSTRACT FROM AUTHOR]