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.

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

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in 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 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant.

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).

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

is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. 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.

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.

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.

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.

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.

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.

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.

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).

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.

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).

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 that associate with methylated DNA.