A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease.

Mitophagy is a fundamental quality control mechanism of mitochondria. Its regulatory mechanisms and pathological implications remain poorly understood. Here, via a mitochondria-targeted genetic screen, we found that knockout (KO) of FBXL4, a mitochondrial disease gene, hyperactivates mitophagy at basal conditions. Subsequent counter screen revealed that FBXL4-KO hyperactivates mitophagy via two mitophagy receptors BNIP3 and NIX. We determined that FBXL4 functions as an integral outer-membrane protein that forms an SCF-FBXL4 ubiquitin E3 ligase complex. SCF-FBXL4 ubiquitinates BNIP3 and NIX to target them for degradation. Pathogenic FBXL4 mutations disrupt SCF-FBXL4 assembly and impair substrate degradation. Fbxl4-/- mice exhibit elevated BNIP3 and NIX proteins, hyperactive mitophagy, and perinatal lethality. Importantly, knockout of either Bnip3 or Nix rescues metabolic derangements and viability of the Fbxl4-/- mice. Together, beyond identifying SCF-FBXL4 as a novel mitochondrial ubiquitin E3 ligase restraining basal mitophagy, our results reveal hyperactivated mitophagy as a cause of mitochondrial disease and suggest therapeutic strategies.

Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast.

The eukaryotic ribosomal RNA (rRNA) is associated cotranscriptionally with numerous factors into an enormous 90S preribosomal particle that conducts early processing of small ribosomal subunits. The assembly pathway and structure of the 90S particle is poorly understood. Here, we affinity-purified and analyzed the constituents of yeast 90S particles that were assembled on a series of plasmid-encoded 3'-truncated pre-18S RNAs. We determined the assembly point of 65 proteins and the U3, U14, and snR30 small nucleolar RNAs (snoRNAs), revealing a stepwise and dynamic assembly map. The 5' external transcribed spacer (ETS) alone can nucleate a large complex. When the 18S rRNA is nearly complete, the 90S structure undergoes a dramatic reorganization, releasing U14, snR30, and 14 protein factors that bind earlier. We also identified a reference state of 90S that is fully assembled yet has not undergone 5'ETS processing. The assembly map present here provides a new framework to understand small subunit biogenesis.

Author Correction: Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells.

Genetically encoded tags for single-molecule imaging in electron microscopy (EM) are long-awaited. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated tags. Next, we exploited this mechanism to develop approaches for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies and mitochondrial matrices. We further implemented cysteine-rich tag-antibody fusion proteins as new immuno-EM probes. Thus, our approaches should allow biologists to address a wide range of biological questions at the single-molecule level in cellular ultrastructural contexts.

PIF3 is phosphorylated by MAPK to modulate plant immunity.

Surface-localized pattern recognition receptors perceive pathogen-associated molecular patterns (PAMPs) to activate pattern-triggered immunity (PTI). Activation of mitogen-activated protein kinases (MAPKs) represents a major PTI response. Here, we report that Arabidopsis thaliana PIF3 negatively regulates plant defense gene expression and resistance to Pseudomonas syringae DC3000. PAMPs trigger phosphorylation of PIF3. Further study reveals that PIF3 interacts with and is phosphorylated by MPK3/6. By mass spectrometry and site-directed mutagenesis, we identified the corresponding phosphorylation sites which fit for SP motif. We further show that a phospho-mimicking PIF3 variant (PIF36D /pifq) conferred increased susceptibility to P. syringae DC3000 and caused lower levels of defense gene expression in plants. Together, this study reveals that PIF3 is phosphorylated by MPK3/6 and phosphorylation of the SP motif residues is required for its negative regulation on plant immunity.

Neurospora COP9 signalosome integrity plays major roles for hyphal growth, conidial development, and circadian function.

The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EX(n)HXHX(10)D) of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCF(FWD-1) complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa.

Multiple PDE3A modulators act as molecular glues promoting PDE3A-SLFN12 interaction and induce SLFN12 dephosphorylation and cell death.

The canonical function of phosphodiesterase 3A (PDE3A) is to hydrolyze the phosphodiester bonds in second messenger molecules, such as cyclic AMP (cAMP) and cyclic guanosine monophosphate (cGMP). Recently, a phosphodiesterase-activity-independent role for PDE3A was reported. In this noncanonical function, PDE3A physically interacts with Schlafen 12 (SLFN12) upon treatment of cells with cytotoxic PDE3A modulators. Here, we confirmed that the cytotoxic PDE3A modulators act as molecular glues to initiate the association of PDE3A and SLFN12. The PDE3A-SLFN12 interaction increases the protein stability of SLFN12 located in the cytoplasm, while at the same time also inducing SLFN12 dephosphorylation (including serines 368 and 573). Mutational analysis demonstrates that dephosphorylation is required for cell death induced by cytotoxic PDE3A modulators. Finally, we found that dephosphorylation promoted the rRNA RNase activity of SLFN12 and show that this nucleolytic activity is essential for SLFN12's cell-death-inducing function. Thus, our study deepens the understanding of the biochemical mechanisms underlying SLFN12-mediated cell death.

A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin2α-induced corpus luteum regression.

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.

Casein kinase 1G2 suppresses necroptosis-promoted testis aging by inhibiting receptor-interacting kinase 3.

Casein kinases are a large family of intracellular serine/threonine kinases that control a variety of cellular signaling functions. Here we report that a member of casein kinase 1 family, casein kinase 1G2, CSNK1G2, binds and inhibits the activation of receptor-interacting kinase 3, RIPK3, thereby attenuating RIPK3-mediated necroptosis. The binding of CSNK1G2 to RIPK3 is triggered by auto-phosphorylation at serine 211/threonine 215 sites in its C-terminal domain. CSNK1G2-knockout mice showed significantly enhanced necroptosis response and premature aging of their testis, a phenotype that was rescued by either double knockout of the Ripk3 gene or feeding the animal with a RIPK1 kinase inhibitor-containing diet. Moreover, CSNK1G2 is also co-expressed with RIPK3 in human testis, and the necroptosis activation marker phospho-MLKL was observed in the testis of old (>80) but not young men, indicating that the testis-aging program carried out by the RIPK3-mediated and CSNK1G2-attenuated necroptosis is evolutionarily conserved between mice and men.

Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration.

Necroptosis, a form of programmed cell death, is characterized by the loss of membrane integrity and release of intracellular contents, the execution of which depends on the membrane-disrupting activity of the Mixed Lineage Kinase Domain-Like protein (MLKL) upon its phosphorylation. Here we found myofibers committed MLKL-dependent necroptosis after muscle injury. Either pharmacological inhibition of the necroptosis upstream kinase Receptor Interacting Protein Kinases 1 (RIPK1) or genetic ablation of MLKL expression in myofibers led to significant muscle regeneration defects. By releasing factors into the muscle stem cell (MuSC) microenvironment, necroptotic myofibers facilitated muscle regeneration. Tenascin-C (TNC), released by necroptotic myofibers, was found to be critical for MuSC proliferation. The temporary expression of TNC in myofibers is tightly controlled by necroptosis; the extracellular release of TNC depends on necroptotic membrane rupture. TNC directly activated EGF receptor (EGFR) signaling pathway in MuSCs through its N-terminus assembly domain together with the EGF-like domain. These findings indicate that necroptosis plays a key role in promoting MuSC proliferation to facilitate muscle regeneration.

Activity-based protein profiling reveals GSTO1 as the covalent target of piperlongumine and a promising target for combination therapy for cancer.

Through systematic target identification for piperlongumine, a cancer-selective killing molecule, we identified GSTO1 as its major covalent target for cancer cell death induction. We also reveal that GSTO1 inhibition is a promising combination strategy with other anti-cancer agents by drug combination screening in which piperlongumine exhibits broad-spectrum synergistic effects with a large proportion of the tested anti-cancer agents, especially with PI3K/Akt/mTOR pathway inhibitors.

Mrg15 stimulates Ash1 H3K36 methyltransferase activity and facilitates Ash1 Trithorax group protein function in Drosophila.

Ash1 is a Trithorax group protein that possesses H3K36-specific histone methyltransferase activity, which antagonizes Polycomb silencing. Here we report the identification of two Ash1 complex subunits, Mrg15 and Nurf55. In vitro, Mrg15 stimulates the enzymatic activity of Ash1. In vivo, Mrg15 is recruited by Ash1 to their common targets, and Mrg15 reinforces Ash1 chromatin association and facilitates the proper deposition of H3K36me2. To dissect the functional role of Mrg15 in the context of the Ash1 complex, we identify an Ash1 point mutation (Ash1-R1288A) that displays a greatly attenuated interaction with Mrg15. Knock-in flies bearing this mutation display multiple homeotic transformation phenotypes, and these phenotypes are partially rescued by overexpressing the Mrg15-Nurf55 fusion protein, which stabilizes the association of Mrg15 with Ash1. In summary, Mrg15 is a subunit of the Ash1 complex, a stimulator of Ash1 enzymatic activity and a critical regulator of the TrxG protein function of Ash1 in Drosophila.

Activation-Dependent Destruction of a Co-receptor by a Pseudomonas syringae Effector Dampens Plant Immunity.

The Arabidopsis immune receptor FLS2 and co-receptor BAK1 perceive the bacterial flagellin epitope flg22 to activate plant immunity. To prevent this response, phytopathogenic bacteria deploy a repertoire of effector proteins to perturb immune signaling. However, the effector-induced perturbation is often sensed by the host, triggering another layer of immunity. We report that the Pseudomonas syringae effector HopB1 acts as a protease to cleave immune-activated BAK1. Prior to activation, HopB1 constitutively interacts with FLS2. Upon activation by flg22, BAK1 is recruited to the FLS2-HopB1 complex and is phosphorylated at Thr455. HopB1 then specifically cleaves BAK1 between Arg297 and Gly298 to inhibit FLS2 signaling. Although perturbation of BAK1 is known to trigger increased immune responses in plants, the HopB1-mediated cleavage of BAK1 leads to enhanced virulence, but not disease resistance. This study thus reveals a virulence strategy by which a pathogen effector attacks the plant immune system with minimal host perturbation.

The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity.

The Arabidopsis immune receptor FLS2 senses the bacterial flagellin epitope flg22 to activate transient elevation of cytosolic calcium ions, production of reactive oxygen species (ROS), and other signaling events to coordinate antimicrobial defenses, such as stomatal closure that limits bacterial invasion. However, how FLS2 regulates these signaling events remains largely unknown. Here we show that the receptor-like cytoplasmic kinase BIK1, a component of the FLS2 immune receptor complex, not only positively regulates flg22-triggered calcium influx but also directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation. Furthermore, BIK1 and RbohD form a pathway that controls stomatal movement in response to flg22, thereby restricting bacterial entry into leaf tissues. These findings highlight a direct role of the FLS2 complex in the regulation of RbohD-mediated ROS production and stomatal defense.