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.

The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis.

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

RGS10 physically and functionally interacts with STIM2 and requires store-operated calcium entry to regulate pro-inflammatory gene expression in microglia.

Chronic activation of microglia is a driving factor in the progression of neuroinflammatory diseases, and mechanisms that regulate microglial inflammatory signaling are potential targets for novel therapeutics. Regulator of G protein Signaling 10 is the most abundant RGS protein in microglia, where it suppresses inflammatory gene expression and reduces microglia-mediated neurotoxicity. In particular, microglial RGS10 downregulates the expression of pro-inflammatory mediators including cyclooxygenase 2 (COX-2) following stimulation with lipopolysaccharide (LPS). However, the mechanism by which RGS10 affects inflammatory signaling is unknown and is independent of its canonical G protein targeted mechanism. Here, we sought to identify non-canonical RGS10 interacting partners that mediate its anti-inflammatory mechanism. Through RGS10 co-immunoprecipitation coupled with mass spectrometry, we identified STIM2, an endoplasmic reticulum (ER) localized calcium sensor and a component of the store-operated calcium entry (SOCE) machinery, as a novel RGS10 interacting protein in microglia. Direct immunoprecipitation experiments confirmed RGS10-STIM2 interaction in multiple microglia and macrophage cell lines, as well as in primary cells, with no interaction observed with the homologue STIM1. We further determined that STIM2, Orai channels, and the calcium-dependent phosphatase calcineurin are essential for LPS-induced COX-2 production in microglia, and this pathway is required for the inhibitory effect of RGS10 on COX-2. Additionally, our data demonstrated that RGS10 suppresses SOCE triggered by ER calcium depletion and that ER calcium depletion, which induces SOCE, amplifies pro-inflammatory genes. In addition to COX-2, we also show that RGS10 suppresses the expression of pro-inflammatory cytokines in microglia in response to thrombin and LPS stimulation, and all of these effects require SOCE. Collectively, the physical and functional links between RGS10 and STIM2 suggest a complex regulatory network connecting RGS10, SOCE, and pro-inflammatory gene expression in microglia, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.

APEX2 Proximity Proteomics Resolves Flagellum Subdomains and Identifies Flagellum Tip-Specific Proteins in Trypanosoma brucei.

Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum (cilium) that plays critical roles in transmission and pathogenesis. An emerging concept is that the flagellum is organized into subdomains, each having specialized composition and function. The overall flagellum proteome has been well studied, but a critical knowledge gap is the protein composition of individual subdomains. We have tested whether APEX-based proximity proteomics could be used to examine the protein composition of T. brucei flagellum subdomains. As APEX-based labeling has not previously been described in T. brucei, we first fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, a well-characterized axonemal complex. We found that DRC1-APEX2 directs flagellum-specific biotinylation, and purification of biotinylated proteins yields a DRC1 "proximity proteome" having good overlap with published proteomes obtained from purified axonemes. Having validated the use of APEX2 in T. brucei, we next attempted to distinguish flagellar subdomains by fusing APEX2 to a flagellar membrane protein that is restricted to the flagellum tip, AC1, and another one that is excluded from the tip, FS179. Fluorescence microscopy demonstrated subdomain-specific biotinylation, and principal-component analysis showed distinct profiles between AC1-APEX2 and FS179-APEX2. Comparing these two profiles allowed us to identify an AC1 proximity proteome that is enriched for tip proteins, including proteins involved in signaling. Our results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve the proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCE Sleeping sickness is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need for approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei and has allowed identification of proximity proteomes for different flagellar subdomains that cannot be purified. This capacity opens the possibility to study the composition and function of other compartments. We expect this approach may be extended to other eukaryotic pathogens and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilium function is impaired.

Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides.

MicroRNAs (miRNAs) function cell-intrinsically to regulate gene expression by base-pairing to complementary mRNA targets while in association with Argonaute, the effector protein of the miRNA-mediated silencing complex (miRISC). A relatively dilute population of miRNAs can be found extracellularly in body fluids such as human blood plasma and cerebrospinal fluid (CSF). The remarkable stability of circulating miRNAs in such harsh extracellular environments can be attributed to their association with protective macromolecular complexes, including extracellular vesicles (EVs), proteins such as Argonaut 2 (AGO2), or high-density lipoproteins. The precise origins and the potential biological significance of various forms of miRNA-containing extracellular complexes are poorly understood. It is also not known whether extracellular miRNAs in their native state may retain the capacity for miRISC-mediated target RNA binding. To explore the potential functionality of circulating extracellular miRNAs, we comprehensively investigated the association between circulating miRNAs and the miRISC Argonaute AGO2. Using AGO2 immunoprecipitation (IP) followed by small-RNA sequencing, we find that miRNAs in circulation are primarily associated with antibody-accessible miRISC/AGO2 complexes. Moreover, we show that circulating miRNAs can base-pair with a target mimic in a seed-based manner, and that the target-bound AGO2 can be recovered from blood plasma in an ∼1:1 ratio with the respective miRNA. Our findings suggest that miRNAs in circulation are largely contained in functional miRISC/AGO2 complexes under normal physiological conditions. However, we find that, in human CSF, the assortment of certain extracellular miRNAs into free miRISC/AGO2 complexes can be affected by pathological conditions such as amyotrophic lateral sclerosis.

NAP1-RELATED PROTEIN1 and 2 negatively regulate H2A.Z abundance in chromatin in Arabidopsis.

In eukaryotes, DNA wraps around histones to form nucleosomes, which are compacted into chromatin. DNA-templated processes, including transcription, require chromatin disassembly and reassembly mediated by histone chaperones. Additionally, distinct histone variants can replace core histones to regulate chromatin structure and function. Although replacement of H2A with the evolutionarily conserved H2A.Z via the SWR1 histone chaperone complex has been extensively studied, in plants little is known about how a reduction of H2A.Z levels can be achieved. Here, we show that NRP proteins cause a decrease of H2A.Z-containing nucleosomes in Arabidopsis under standard growing conditions. nrp1-1 nrp2-2 double mutants show an over-accumulation of H2A.Z genome-wide, especially at heterochromatic regions normally H2A.Z-depleted in wild-type plants. Our work suggests that NRP proteins regulate gene expression by counteracting SWR1, thereby preventing excessive accumulation of H2A.Z.

Proximity biotinylation reveals novel secreted dense granule proteins of Toxoplasma gondii bradyzoites.

Toxoplasma gondii is an obligate intracellular parasite which is capable of establishing life-long chronic infection in any mammalian host. During the intracellular life cycle, the parasite secretes an array of proteins into the parasitophorous vacuole (PV) where it resides. Specialized organelles called the dense granules secrete GRA proteins that are known to participate in nutrient acquisition, immune evasion, and host cell-cycle manipulation. Although many GRAs have been discovered which are expressed during the acute infection mediated by tachyzoites, little is known about those that participate in the chronic infection mediated by the bradyzoite form of the parasite. In this study, we sought to uncover novel bradyzoite-upregulated GRA proteins using proximity biotinylation, which we previously used to examine the secreted proteome of the tachyzoites. Using a fusion of the bradyzoite upregulated protein MAG1 to BirA* as bait and a strain with improved switch efficiency, we identified a number of novel GRA proteins which are expressed in bradyzoites. After using the CRISPR/Cas9 system to characterize these proteins by gene knockout, we focused on one of these GRAs (GRA55) and found it was important for the establishment or maintenance of cysts in the mouse brain. These findings highlight new components of the GRA proteome of the tissue-cyst life stage of T. gondii and identify potential targets that are important for maintenance of parasite persistence in vivo.

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.

Using BioID for the Identification of Interacting and Proximal Proteins in Subcellular Compartments in Toxoplasma gondii.

BioID is an in vivo biotinylation system developed to examine the proximal and interacting proteins of a bait protein within a subcellular compartment. This approach has been exploited in Toxoplasma for protein-protein interaction studies and proteomic characterizations of intracellular compartments. The BioID method requires constructing a translational fusion between a protein of interest and the promiscuous biotin ligase BirA∗ (a mutant of the E. coli protein BirA) which enables trafficking of the protein to the correct intracellular compartment and association with its partners. Proximity labelling occurs upon addition of biotin to the media and the biotinylated target proteins are then be purified using stringent conditions via streptavidin chromatography. In this chapter, we describe the methodology to fuse BirA∗ (or the newer variant BioID2) to a bait protein using endogenous gene tagging in Toxoplasma and then identify the proximal and interacting proteins using in vivo biotinylation, streptavidin purification and mass spectrometric analysis.

Chemical Derivatization of Affinity Matrices Provides Protection from Tryptic Proteolysis.

The enrichment of biotinylated proteins using immobilized streptavidin has become a staple methodology for affinity purification-based proteomics. Many of these workflows rely upon tryptic digestion to elute streptavidin-captured moieties from the beads. The concurrent release of high amounts of streptavidin-derived peptides into the digested sample, however, can significantly hamper the effectiveness of downstream proteomic analyses by increasing the complexity and dynamic range of the mixture. Here, we describe a strategy for the chemical derivatization of streptavidin that renders it largely resistant to proteolysis by trypsin and thereby dramatically reduces the amount of streptavidin contamination in the sample. This rapid and robust approach improves the effectiveness of mass spectrometry-based characterization of streptavidin-purified samples making it broadly useful for a wide variety of applications. In addition, we show that this chemical protection strategy can also be applied to other affinity matrices including immobilized antibodies against HA epitopes.

Arabidopsis SWR1-associated protein methyl-CpG-binding domain 9 is required for histone H2A.Z deposition.

Deposition of the histone variant H2A.Z by the SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation in eukaryotes, but the composition of the Arabidopsis SWR1-C has not been thoroughly characterized. Here, we aim to identify interacting partners of a conserved Arabidopsis SWR1 subunit ACTIN-RELATED PROTEIN 6 (ARP6). We isolate nine predicted components and identify additional interactors implicated in histone acetylation and chromatin biology. One of the interacting partners, methyl-CpG-binding domain 9 (MBD9), also strongly interacts with the Imitation SWItch (ISWI) chromatin remodeling complex. MBD9 is required for deposition of H2A.Z at a distinct subset of ARP6-dependent loci. MBD9 is preferentially bound to nucleosome-depleted regions at the 5' ends of genes containing high levels of activating histone marks. These data suggest that MBD9 is a SWR1-C interacting protein required for H2A.Z deposition at a subset of actively transcribing genes.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

Sequential Windowed Acquisition of Reporter Masses for Quantitation-First Proteomics.

The standard approach for proteomic data acquisition of isobaric-tagged samples by mass spectrometry is data-dependent acquisition. This semistochastic, identification-first paradigm generates a wealth of peptide-level data without regard to relative abundance. We introduce a data acquisition concept called sequential windowed acquisition of reporter masses (SWARM). This approach performs quantitation first, thereby allowing subsequent acquisition decisions to be predicated on user-defined patterns of reporter ion intensities. The efficacy of this approach is validated through experiments with both synthetic mixtures of Escherichia coli ribosomes spiked into human cell lysates at known ratios and the quantitative evaluation of the human proteome's response to the inhibition of cullin-based protein ubiquitination via the small molecule MLN4924. We find that SWARM-informed parallel reaction monitoring acquisitions display effective acquisition biasing toward analytes displaying quantitative characteristics of interest, resulting in an improvement in the detection of differentially abundant analytes. The SWARM concept provides a flexible platform for the further development of new acquisition methods.

A DNA methylation reader complex that enhances gene transcription.

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.