Wild-type C-Raf gene dosage and dimerization drive prostate cancer metastasis.

Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf's ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

A synthetic coolant (WS-23) in disposable electronic cigarettes impairs cytoskeletal function in EpiAirway microtissues exposed at the air liquid interface.

The design of popular disposable electronic cigarettes (ECs) was analyzed, and the concentrations of WS-23, a synthetic coolant, in EC fluids were determined for 22 devices from 4 different brands. All products contained WS-23 in concentrations that ranged from 1.0 to 40.1 mg/mL (mean = 21.4 ± 9.2 mg/mL). To determine the effects of WS-23 on human bronchial epithelium in isolation of other chemicals, we exposed EpiAirway 3-D microtissues to WS-23 at the air liquid interface (ALI) using a cloud chamber that generated aerosols without heating. Proteomics analysis of exposed tissues revealed that the cytoskeleton was a major target of WS-23. BEAS-2B cells were exposed to WS-23 in submerged culture to validate the main results from proteomics. F-actin, which was visualized with phalloidin, decreased concentration dependently in WS-23 treated BEAS-2B cells, and cells became immotile in concentrations above 1.5 mg/mL. Gap closure, which depends on both cell proliferation and migration, was inhibited by 0.45 mg/mL of WS-23. These data show that WS-23 is being added to popular EC fluids at concentrations that can impair processes dependent on the actin cytoskeleton and disturb homeostasis of the bronchial epithelium. The unregulated use of WS-23 in EC products may harm human health.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma's ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with Δ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon gamma (IFN-γ) in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-κB complex. While GRA15 similarly regulates NF-κB, infection with Δ gra83/ Δ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. Importance: Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

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.

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.

Human Multisubunit E3 Ubiquitin Ligase Required for Heterotrimeric G-Protein ß-Subunit Ubiquitination and Downstream Signaling.

G-protein-coupled receptors (GPCRs) initiate intracellular signaling events through heterotrimeric G-protein α-subunits (Gα) and the ßγ-subunit dimer (Gßγ). In this study, we utilized mass spectrometry to identify novel regulators of Gßγ signaling in human cells. This prompted our characterization of KCTD2 and KCTD5, two related potassium channel tetramerization domain (KCTD) proteins that specifically recognize Gßγ. We demonstrated that these KCTD proteins are substrate adaptors for a multisubunit CUL3-RING ubiquitin ligase, in which a KCTD2-KCTD5 hetero-oligomer associates with CUL3 through KCTD5 subunits and recruits Gßγ through both KCTD proteins in response to G-protein activation. These KCTD proteins promote monoubiquitination of lysine-23 within Gß1/2in vitro and in HEK-293 cells. Depletion of these adaptors from cancer cell lines sharply impairs downstream signaling. Together, our studies suggest that a KCTD2-KCTD5-CUL3-RING E3 ligase recruits Gßγ in response to signaling, monoubiquitinates lysine-23 within Gß1/2, and regulates Gßγ effectors to modulate downstream signal transduction.

Arabidopsis MORC proteins function in the efficient establishment of RNA directed DNA methylation.

The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms.

Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics.

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of coeluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias toward high-abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or carboxylate-modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange and reversed phase-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH reversed phase. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low-abundance bait proteins.