The SARS coronavirus accessory protein ORF3a rescues potassium conductance in yeast.

ORF3a is an accessory protein expressed by all human pathogen coronaviruses and is the only accessory protein that strongly affects viral fitness. Its deletion reduces severity in both alpha- and beta-coronaviruses, demonstrating a conserved function across the superfamily. Initially regarded as a non-selective cation channel, ORF3a's function is now disputed. Here, we show that ORF3a from SARS, but not SARS-CoV-2, promotes potassium conductance in a yeast model system commonly used to study potassium channels. ORF3a-mediated potassium conductance is also sensitive to inhibitors, including emodin, carbamazepine, and nifedipine. This model may be used in future studies on ORF3a and related proteins.

Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis.

After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in predicted enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.

TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs.

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+-activated Cl- channel TMEM16A. To do so, fertilization activates phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC during the fast block remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLCγ and PLCß. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLCγ, or inhibitors of Gαq/11 pathways, used to stop activation of PLCß. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.

Xenopus laevis lack the critical sperm factor PLCζ.

Fertilization of eggs from the African clawed frog Xenopus laevis is characterized by an increase in cytosolic calcium, a phenomenon that is also observed in other vertebrates such as mammals and birds. During fertilization in mammals and birds, the transfer of the soluble PLCζ from sperm into the egg is thought to trigger the release of calcium from the endoplasmic reticulum (ER). Injecting sperm extracts into eggs reproduces this effect, reinforcing the hypothesis that a sperm factor is responsible for calcium release and egg activation. Remarkably, this occurs even when sperm extracts from X. laevis are injected into mouse eggs, suggesting that mammals and X. laevis share a sperm factor. However, X. laevis lacks an annotated PLCZ1 gene, which encodes the PLCζ enzyme. In this study, we attempted to determine whether sperm from X. laevis express an unannotated PLCZ1 ortholog. We identified PLCZ1 orthologs in 11 amphibian species, including 5 that had not been previously characterized, but did not find any in either X. laevis or the closely related Xenopus tropicalis. Additionally, we performed RNA sequencing on testes obtained from adult X. laevis males and did not identify potential PLCZ1 orthologs in our dataset or in previously collected ones. These findings suggest that PLCZ1 may have been lost in the Xenopus lineage and raise the question of how fertilization triggers calcium release and egg activation in these species.

Phosphate position is key in mediating transmembrane ion channel TMEM16A-phosphatidylinositol 4,5-bisphosphate interaction.

TransMEMbrane 16A (TMEM16A) is a Ca2+-activated Cl- channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P2 head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca2+ to excised inside-out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P2 analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P2 reduces TMEM16A-conducted currents. Application of PIP2 analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4' position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P2 binds to and potentiates TMEM16A channels.

Inactivation of RIP3 kinase sensitizes to 15LOX/PEBP1-mediated ferroptotic death.

Ferroptosis and necroptosis are two pro-inflammatory cell death programs contributing to major pathologies and their inhibition has gained attention to treat a wide range of disease states. Necroptosis relies on activation of RIP1 and RIP3 kinases. Ferroptosis is triggered by oxidation of polyunsaturated phosphatidylethanolamines (PUFA-PE) by complexes of 15-Lipoxygenase (15LOX) with phosphatidylethanolamine-binding protein 1 (PEBP1). The latter, also known as RAF kinase inhibitory protein, displays promiscuity towards multiple proteins. In this study we show that RIP3 K51A kinase inactive mice have increased ferroptotic burden and worse outcome after irradiation and brain trauma rescued by anti-ferroptotic compounds Liproxstatin-1 and Ferrostatin 16-86. Given structural homology between RAF and RIP3, we hypothesized that PEBP1 acts as a necroptosis-to-ferroptosis switch interacting with either RIP3 or 15LOX. Using genetic, biochemical, redox lipidomics and computational approaches, we uncovered that PEBP1 complexes with RIP3 and inhibits necroptosis. Elevated expression combined with higher affinity enables 15LOX to pilfer PEBP1 from RIP3, thereby promoting PUFA-PE oxidation and ferroptosis which sensitizes Rip3K51A/K51A kinase-deficient mice to total body irradiation and brain trauma. This newly unearthed PEBP1/15LOX-driven mechanism, along with previously established switch between necroptosis and apoptosis, can serve multiple and diverse cell death regulatory functions across various human disease states.

PEBP1 acts as a rheostat between prosurvival autophagy and ferroptotic death in asthmatic epithelial cells.

Temporally harmonized elimination of damaged or unnecessary organelles and cells is a prerequisite of health. Under Type 2 inflammatory conditions, human airway epithelial cells (HAECs) generate proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) as proximate death signals. Production of 15-HpETE-PE depends on activation of 15-lipoxygenase-1 (15LO1) in complex with PE-binding protein-1 (PEBP1). We hypothesized that cellular membrane damage induced by these proferroptotic phospholipids triggers compensatory prosurvival pathways, and in particular autophagic pathways, to prevent cell elimination through programmed death. We discovered that PEBP1 is pivotal to driving dynamic interactions with both proferroptotic 15LO1 and the autophagic protein microtubule-associated light chain-3 (LC3). Further, the 15LO1-PEBP1-generated ferroptotic phospholipid, 15-HpETE-PE, promoted LC3-I lipidation to stimulate autophagy. This concurrent activation of autophagy protects cells from ferroptotic death and release of mitochondrial DNA. Similar findings are observed in Type 2 Hi asthma, where high levels of both 15LO1-PEBP1 and LC3-II are seen in HAECs, in association with low bronchoalveolar lavage fluid mitochondrial DNA and more severe disease. The concomitant activation of ferroptosis and autophagy by 15LO1-PEBP1 complexes and their hydroperoxy-phospholipids reveals a pathobiologic pathway relevant to asthma and amenable to therapeutic targeting.

The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway.

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance.

Empowerment of 15-Lipoxygenase Catalytic Competence in Selective Oxidation of Membrane ETE-PE to Ferroptotic Death Signals, HpETE-PE.

sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines ( sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of ∼100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals.

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

Comparative Analysis of Ciliary Membranes and Ectosomes.

Primary and motile cilia/flagella function as cellular antennae, receiving signals from the environment and subsequently activating signaling pathways that are critical for cellular homeostasis and differentiation [1-3]. Recent work with the green alga Chlamydomonas and the nematode C. elegans demonstrated that ectosomes can be released from the cilium and can mediate the intercellular communication [4-9]. To better understand the function of flagellar ectosomes, we have compared their protein composition to that of the flagellar membrane from which they are derived. Ectosomes released from flagella have a unique protein composition, being enriched in a subset of flagellar membrane proteins, proteases, proteins from the endosomal sorting complex required for transport (ESCRT) [10-12], small GTPases, and ubiquitinated proteins. Live imaging showed that an ESCRT-related protein (PDCD6) was enriched in ectosomes released from flagella during gamete activation. We devised a sensitive and rapid assay to monitor ectosome release using luciferase fused to PDCD6 and a mutated ubiquitin. Ectosome release increased when cells underwent flagellar resorption. Knockdown of two ESCRT-related proteins, PDCD6 and VPS4, attenuated ectosome release during flagellar shortening and shortening was slowed. These data suggest that the ESCRT proteins mediate ectosome release and thereby influence flagellar shortening in Chlamydomonas. In addition, the prevalence of receptors such as agglutinin and ubiquitinated proteins in ciliary ectosomes suggests that they are involved in cell signaling and turnover of ciliary proteins.

Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1.

Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph(+)B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GRB2-associated binding protein 2 (GAB2) as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transducedGab2(-/-)bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors phosphatidylinositol 3-kinase (PI3K) or SRC homology 2-containing phosphotyrosine phosphatase 2 (SHP2). GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph(+)hematologic neoplasms.

A Mechanism of Subunit Recruitment in Human Small Heat Shock Protein Oligomers.

Small heat shock proteins (sHSPs) make up a class of molecular chaperones broadly observed across organisms. Many sHSPs form large oligomers that undergo dynamic subunit exchange that is thought to play a role in chaperone function. Though remarkably heterogeneous, sHSP oligomers share three types of intermolecular interactions that involve all three defined regions of a sHSP: the N-terminal region (NTR), the conserved α-crystallin domain (ACD), and a C-terminal region (CTR). Here we define the structural interactions involved in incorporation of a subunit into a sHSP oligomer. We demonstrate that a minimal ACD dimer of the human sHSP, HSPB5, interacts with an HSPB5 oligomer through two types of interactions: (1) interactions with CTRs in the oligomer and (2) via exchange into and out of the dimer interface composed of two ACDs. Unexpectedly, although dimers are thought to be the fundamental building block for sHSP oligomers, our results clearly indicate that subunit exchange into and out of oligomers occurs via monomers. Using structure-based mutants, we show that incorporation of a subunit into an oligomer is predicated on recruitment of the subunit via its interaction with CTRs on an oligomer. Both the rate and extent of subunit incorporation depend on the accessibility of CTRs within an HSPB5 oligomer. We show that this mechanism also applies to formation of heterooligomeric sHSP species composed of HSPB5 and HSPB6 and is likely general among sHSPs. Finally, our observations highlight the importance of NTRs in the thermodynamic stability of sHSP oligomers.

Structural mechanism for the regulation of HCN ion channels by the accessory protein TRIP8b.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels underlie the cationic Ih current present in many neurons. The direct binding of cyclic AMP to HCN channels increases the rate and extent of channel opening and results in a depolarizing shift in the voltage dependence of activation. TRIP8b is an accessory protein that regulates the cell surface expression and dendritic localization of HCN channels and reduces the cyclic nucleotide dependence of these channels. Here, we use electron paramagnetic resonance (EPR) to show that TRIP8b binds to the apo state of the cyclic nucleotide binding domain (CNBD) of HCN2 channels without changing the overall domain structure. With EPR and nuclear magnetic resonance, we locate TRIP8b relative to the HCN channel and identify the binding interface on the CNBD. These data provide a structural framework for understanding how TRIP8b regulates the cyclic nucleotide dependence of HCN channels.

Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins.

The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane [1]. Located near the base of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence [2-6]. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia-related diseases, or ciliopathies, presenting symptoms including renal cysts, retinal degeneration, and situs inversus [7-9]. Some TZ proteins have been identified and shown to interact with each other through coprecipitation studies in vertebrate cells [4, 10, 11] and genetics studies in C. elegans [3]. As a distinct approach to identify TZ proteins, we have taken advantage of the biology of Chlamydomonas to isolate TZs. Proteomic analysis identified 115 proteins, ten of which were known TZ proteins related to ciliopathies, indicating that the preparation was highly enriched for TZs. Interestingly, six proteins of the endosomal sorting complexes required for transport (ESCRT) were also associated with the TZs. Identification of these and other proteins in the TZ will provide new insights into functions of the TZ, as well as candidate ciliopathy genes.

Ciliary ectosomes: transmissions from the cell's antenna.

The cilium is the site of function for a variety of membrane receptors, enzymes and signal transduction modules crucial for a spectrum of cellular processes. Through targeted transport and selective gating mechanisms, the cell localizes specific proteins to the cilium that equip it for the role of sensory antenna. This capacity of the cilium to serve as a specialized compartment where specific proteins can be readily concentrated for sensory reception also makes it an ideal organelle to employ for the regulated emission of specific biological material and information. In this review we present and discuss an emerging body of evidence centered on ciliary ectosomes - bioactive vesicles released from the surface of the cilium.

Proteins of the ciliary axoneme are found on cytoplasmic membrane vesicles during growth of cilia.

The cilium is a specialized extension of the cell in which many specific proteins are admitted and retained, while many others are excluded or expelled. In order to maintain the organelle, the cell must possess mechanisms for the selective gating of protein entry, as well as for the targeted transport of proteins to the cilium from their sites of synthesis within the cell [1-4]. We hypothesized that the cell employs cytoplasmic vesicles as vehicles not only for the transport of proteins destined for the ciliary membrane but also for the transport of axonemal proteins to the cilium by means of peripheral association with vesicles. To test this hypothesis, we employed two different experimental strategies: (1) isolation and biochemical characterization of cytoplasmic vesicles that carry ciliary proteins, and (2) in situ localization of ciliary proteins on cytoplasmic vesicle surfaces using gold labeling and electron microscopy. Our findings indicate that structural proteins destined for the ciliary axoneme are attached to the outer surfaces of cytoplasmic vesicles that carry integral ciliary membrane proteins during the process of ciliary growth.

Flavonoid regulation of HCN2 channels.

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC50) of 1.8 µM. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.

Centrosomal protein CEP104 (Chlamydomonas FAP256) moves to the ciliary tip during ciliary assembly.

The ciliary tip has been implicated in ciliary assembly and disassembly, and signaling, yet information on its protein composition is limited. Using comparative, quantitative proteomics based on the fact that tip proteins will be approximately twice as concentrated in half-length compared with full-length flagella, we have identified FAP256 as a tip protein in Chlamydomonas. FAP256 localizes to the tips of both central pair and outer doublet microtubules (MTs) and it remains at the tip during flagellar assembly and disassembly. Similarly, its vertebrate counterpart, CEP104, localizes on the distal ends of both centrioles of nondividing cells until the mother centriole forms a cilium and then localizes at the tip of the elongating cilium. A null mutant of FAP256 in Chlamydomonas and RNAi in vertebrate cells showed that FAP256/CEP104 is required for ciliogenesis in a high percentage of cells. In those cells that could form cilia, there were structural deformities at the ciliary tips.