The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

Crym-positive striatal astrocytes gate perseverative behaviour.

Astrocytes are heterogeneous glial cells of the central nervous system1-3. However, the physiological relevance of astrocyte diversity for neural circuits and behaviour remains unclear. Here we show that a specific population of astrocytes in the central striatum expresses µ-crystallin (encoded by Crym in mice and CRYM in humans) that is associated with several human diseases, including neuropsychiatric disorders4-7. In adult mice, reducing the levels of µ-crystallin in striatal astrocytes through CRISPR-Cas9-mediated knockout of Crym resulted in perseverative behaviours, increased fast synaptic excitation in medium spiny neurons and dysfunctional excitatory-inhibitory synaptic balance. Increased perseveration stemmed from the loss of astrocyte-gated control of neurotransmitter release from presynaptic terminals of orbitofrontal cortex-striatum projections. We found that perseveration could be remedied using presynaptic inhibitory chemogenetics8, and that this treatment also corrected the synaptic deficits. Together, our findings reveal converging molecular, synaptic, circuit and behavioural mechanisms by which a molecularly defined and allocated population of striatal astrocytes gates perseveration phenotypes that accompany neuropsychiatric disorders9-12. Our data show that Crym-positive striatal astrocytes have key biological functions within the central nervous system, and uncover astrocyte-neuron interaction mechanisms that could be targeted in treatments for perseveration.

MYCT1 controls environmental sensing in human haematopoietic stem cells.

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

Astrocyte-neuron subproteomes and obsessive-compulsive disorder mechanisms.

Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their respective contributions to physiology and disease. Here we used cell-specific and subcompartment-specific proximity-dependent biotinylation1 to study the proteomes of striatal astrocytes and neurons in vivo. We evaluated cytosolic and plasma membrane compartments for astrocytes and neurons to discover how these cells differ at the protein level in their signalling machinery. We also assessed subcellular compartments of astrocytes, including end feet and fine processes, to reveal their subproteomes and the molecular basis of essential astrocyte signalling and homeostatic functions. Notably, SAPAP3 (encoded by Dlgap3), which is associated with obsessive-compulsive disorder (OCD) and repetitive behaviours2-8, was detected at high levels in striatal astrocytes and was enriched within specific astrocyte subcompartments where it regulated actin cytoskeleton organization. Furthermore, genetic rescue experiments combined with behavioural analyses and molecular assessments in a mouse model of OCD4 lacking SAPAP3 revealed distinct contributions of astrocytic and neuronal SAPAP3 to repetitive and anxiety-related OCD-like phenotypes. Our data define how astrocytes and neurons differ at the protein level and in their major signalling pathways. Moreover, they reveal how astrocyte subproteomes vary between physiological subcompartments and how both astrocyte and neuronal SAPAP3 mechanisms contribute to OCD phenotypes in mice. Our data indicate that therapeutic strategies that target both astrocytes and neurons may be useful to explore in OCD and potentially other brain disorders.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Direct tests of cytochrome c and c1 functions in the electron transport chain of malaria parasites.

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome (cyt) functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knockout had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.

Elucidation of TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes.

The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), nucleoside synthesis (NME1), and mRNA translation and stability (PABPC4). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1 and PABPC4. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in modulating diverse biological processes.

The TRIPLE PHD FINGERS proteins are required for SWI/SNF complex-mediated +1 nucleosome positioning and transcription start site determination in Arabidopsis.

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5'-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5' transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination.

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

FAM111A induces nuclear dysfunction in disease and viral restriction.

Mutations in the nuclear trypsin-like serine protease FAM111A cause Kenny-Caffey syndrome (KCS2) with hypoparathyroidism and skeletal dysplasia or perinatally lethal osteocraniostenosis (OCS). In addition, FAM111A was identified as a restriction factor for certain host range mutants of the SV40 polyomavirus and VACV orthopoxvirus. However, because FAM111A function is poorly characterized, its roles in restricting viral replication and the etiology of KCS2 and OCS remain undefined. We find that FAM111A KCS2 and OCS patient mutants are hyperactive and cytotoxic, inducing apoptosis-like phenotypes such as disruption of nuclear structure and pore distribution, in a protease-dependent manner. Moreover, wild-type FAM111A activity causes similar nuclear phenotypes, including the loss of nuclear barrier function, when SV40 host range mutants attempt to replicate in restrictive cells. Interestingly, pan-caspase inhibitors do not block these FAM111A-induced phenotypes, implying it acts independently or upstream of caspases. In this regard, we identify nucleoporins and the associated GANP transcription/replication factor as FAM111A interactors and candidate targets. Overall, we reveal a potentially unifying mechanism through which deregulated FAM111A activity restricts viral replication and causes KCS2 and OCS.

The plant mobile domain proteins MAIN and MAIL1 interact with the phosphatase PP7L to regulate gene expression and silence transposable elements in Arabidopsis thaliana.

Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs.

Aim32 is a dual-localized 2Fe-2S mitochondrial protein that functions in redox quality control.

Yeast is a facultative anaerobe and uses diverse electron acceptors to maintain redox-regulated import of cysteine-rich precursors via the mitochondrial intermembrane space assembly (MIA) pathway. With the growing diversity of substrates utilizing the MIA pathway, understanding the capacity of the intermembrane space (IMS) to handle different types of stress is crucial. We used MS to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Altered inheritance of mitochondria 32 (Aim32), a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1-binding protein. Detailed localization studies showed that Aim32 resided in both the mitochondrial matrix and IMS. Aim32 interacted with additional proteins including redox protein Osm1 and protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or mutation of conserved cysteine residues that coordinate the Fe-S center in Aim32 resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased. Aim32 also bound to several mitochondrial proteins under nonreducing conditions, suggesting a function in maintaining the redox status of proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress such as elevated temperature and hydroxyurea, and under anaerobic conditions. These studies suggest that the Fe-S protein Aim32 has a potential role in general redox homeostasis in the matrix and IMS. Thus, Aim32 may be poised as a sensor or regulator in quality control for a broad range of mitochondrial proteins.

High-Field Asymmetric Waveform Ion Mobility Spectrometry Interface Enhances Parallel Reaction Monitoring on an Orbitrap Mass Spectrometer.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables gas-phase separations on a chromatographic time scale and has become a useful tool for proteomic applications. Despite its emerging utility, however, the molecular determinants underlying peptide separation by FAIMS have not been systematically investigated. Here, we characterize peptide transmission in a FAIMS device across a broad range of compensation voltages (CVs) and used machine learning to identify charge state and three-dimensional (3D) electrostatic peptide potential as major contributors to peptide intensity at a given CV. We also demonstrate that the machine learning model can be used to predict optimized CV values for peptides, which significantly improves parallel reaction monitoring workflows. Together, these data provide insight into peptide separation by FAIMS and highlight its utility in targeted proteomic applications.

A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex.

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.

SP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome*.

Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-preparation workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample-preparation (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with on-line high-field asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides, we achieve unprecedented coverage of >14000 unique cysteines in a single-shot 70 min experiment. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-molecule screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our analysis of 18 samples from seven cell lines identified 34225 unique cysteines using only ∼28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the technical foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole.

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane. Although the PVM is the site of several transport activities essential to parasite survival, the basis for organisation of this membrane system is unknown. Here, we performed proximity labeling at the PVM with BioID2, which highlighted a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. Importantly, although EXP1 was required for intraerythrocytic development, a previously reported in vitro glutathione S-transferase activity could not account for this essential EXP1 function in vivo. EXP1 knockdown was accompanied by profound changes in vacuole ultrastructure, including apparent increased separation of the PVM from the parasite plasma membrane and formation of abnormal membrane structures. Furthermore, although activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 but not the HSP101 unfoldase was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper organisation of EXP2 within the PVM.

Endo or Exo? Structures of Gas-Phase Alkali Metal Cation/Aromatic Half-Belt Complexes.

1,1,9,9-Tetramethyl[9](2,11)teropyrenophane (TM9TP), a belt-shaped molecule, has a sizable cavity that molecules or ions could occupy. In this study, the question of whether TM9TP forms gas-phase ion-molecule complexes with metal cations (K+ , Rb+ , Cs+ ) situated inside or outside the TM9TP cavity was addressed using both experimental and computational methods. Complexes were trapped in a Fourier transform ion cyclotron resonance mass spectrometer and their structures were explored by some novel physical chemistry/mass spectrometry methods. Blackbody infrared radiative dissociation kinetics reveal two populations of ions, a fast dissociating fraction and a persistent fraction. Infrared multiphoton dissociation spectra (vibrational spectra) provide very strong evidence that the most abundant population is a complex where the metal cation is inside the TM9TP cavity, endo-TM9TP. Red-shifted C-H stretching bands present in the gas-phase vibrational spectra of these ionic complexes show that there is an interaction between the metal cation and bridge C-H bonds due to the cation sitting inside the cavity of TM9TP. B3LYP/6-31+G(d,p) calculations showed the endo complexes to be the lowest in energy; about 60 kJ mol-1 more thermodynamically stable and more than 120 kJ mol-1 kinetically more stable than the exo complex.

Structures and unimolecular chemistry of M(Pro2-H)(+) (M = Mg, Ca, Sr, Ba, Mn, Fe, Co, Ni, Cu, Zn) by IRMPD spectroscopy, SORI-CID, and theoretical studies.

M(Pro2-H)(+) complexes were electrosprayed and isolated in an FTICR cell where their unimolecular chemistries and structures were explored using SORI-CID and IRMPD spectroscopy. These experiments were augmented by computational methods such as electronic structure, simulated annealing, and atoms in molecules (AIM) calculations. The unimolecular chemistries of the larger metal cation (Ca(2+), Sr(2+) and Ba(2+)) complexes predominantly involve loss of neutral proline whereas the complexes involving the smaller Mg(2+) and transition metal dications tend to lose small neutral molecules such as water and carbon dioxide. Interestingly, all complexes involving transition metal dications except for Cu(Pro2-H)(+) lose H2 upon collisional or IRMPD activation. IRMPD spectroscopy shows that the intact proline in the transition metal complexes and Cu(Pro2-H)(+) is predominantly canonical (charge solvated) while for the Ca(2+), Sr(2+), and Ba(2+) complexes, proline is in its zwitterionic form. The IRMPD spectra for both Mg(Pro2-H)(+) and Mn(Pro2-H)(+) are concluded to have contributions from both charge-solvated and canonical structures.

Self-Assembled Multimetallic/Peptide Complexes: Structures and Unimolecular Reactions of [Mn (GlyGly-H)2n-1 ](+) and Mn+1 (GlyGly-H2n ](2+) Clusters in the Gas Phase.

The unimolecular chemistry and structures of self-assembled complexes containing multiple alkaline-earth-metal dications and deprotonated GlyGly ligands are investigated. Singly and doubly charged ions [Mn (GlyGly-H)n-1 ](+) (n=2-4), [Mn+1 (GlyGly-H)2n ](2+) (n=2,4,6), and [M(GlyGly-H)GlyGly](+) were observed. The losses of 132 Da (GlyGly) and 57 Da (determined to be aminoketene) were the major dissociation pathways for singly charged ions. Doubly charged Mg(2+) clusters mainly lost GlyGly, whereas those containing Ca(2+) or Sr(2+) also underwent charge separation. Except for charge separation, no loss of metal cations was observed. Infrared multiple photon dissociation spectra were the most consistent with the computed IR spectra for the lowest energy structures, in which deprotonation occurs at the carboxyl acid groups and all amide and carboxylate oxygen atoms are complexed to the metal cations. The N-H stretch band, observed at 3350 cm(-1) , is indicative of hydrogen bonding between the amine nitrogen atoms and the amide hydrogen atom. This study represents the first into large self-assembled multimetallic complexes bound by peptide ligands.

In vivo identification of astrocyte and neuron subproteomes by proximity-dependent biotinylation.

The central nervous system (CNS) comprises diverse and morphologically complex cells. To understand the molecular basis of their physiology, it is crucial to assess proteins expressed within intact cells. Commonly used methods utilize cell dissociation and sorting to isolate specific cell types such as neurons and astrocytes, the major CNS cells. Proteins purified from isolated cells are identified by mass spectrometry-based proteomics. However, dissociation and cell-sorting methods lead to near total loss of cellular morphology, thereby losing proteins from key relevant subcompartments such as processes, end feet, dendrites and axons. Here we provide a systematic protocol for cell- and subcompartment-specific labeling and identification of proteins found within intact astrocytes and neurons in vivo. This protocol utilizes the proximity-dependent biotinylation system BioID2, selectively expressed in either astrocytes or neurons, to label proximal proteins in a cell-specific manner. BioID2 is targeted genetically to assess the subproteomes of subcellular compartments such as the plasma membrane and sites of cell-cell contacts. We describe in detail the expression methods (variable timing), stereotaxic surgeries for expression (1-2 d and then 3 weeks), in vivo protein labeling (7 d), protein isolation (2-3 d), protein identification methods (2-3 d) and data analysis (1 week). The protocol can be applied to any area of the CNS in mouse models of physiological processes and for disease-related research.