Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity.

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

Excessive intragastric alcohol administration exacerbates hepatic encephalopathy and provokes neuronal cell death in male rats with chronic liver disease.

Hepatic encephalopathy (HE) is defined as decline in neurological function during chronic liver disease (CLD). Alcohol is a major etiological factor in the pathogenesis of fibrosis/cirrhosis and has also been documented to directly impact the brain. However, the role of alcohol in the development of HE in CLD remains unclear. Here, we investigated the impact of excessive alcohol administration on neurological deterioration in rats with CLD. Starting day 7 post-BDL surgery, rats were administered alcohol twice daily (51% v/v ethanol, 3 g/kg, via gavage) for 4 weeks. Motor coordination was assessed weekly using rotarod and anxiety-like behavior was evaluated with open field and elevated plus maze at 5 weeks. Upon sacrifice, brains were collected for western blot and immunohistochemical analyses to investigate neuronal integrity and oxidative stress status. Alcohol worsened motor coordination performance and increased anxiety-like behavior in BDL rats. Impairments were associated with decreased neuronal markers of NeuN and SMI311, increased apoptotic markers of cleaved/pro-caspase-3 and Bax/Bcl2, increased necroptosis markers of pRIP3 and pMLKL, decreased total antioxidant capacity (TAC), and increased 4-hydroxynonenal (4-HNE)modified proteins in the cerebellum of BDL-alcohol rats when compared to respective controls. Immunofluorescence confirmed the colocalization of cleaved caspase-3 and pMLKL in the granular neurons of the cerebellum of BDL-alcohol rats. Excessive alcohol consumption exacerbates HE which leads to associated apoptotic and necroptotic neuronal loss in the cerebellum of BDL-alcohol rats. Additionally, higher levels of 4-HNE and decreased TAC in the cerebellum of BDL-alcohol rats suggest oxidative stress is the triggering factor of apoptotic and necroptotic neuronal loss/injury.

POGZ promotes homology-directed DNA repair in an HP1-dependent manner.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.

HwMR is a novel magnesium-associated protein.

Microbial rhodopsins (MRho) are vital proteins in Haloarchaea for solar light sensing in extreme living environments. Among them, Haloquadratum walsbyi (Hw) is a species known to survive high MgCl2 concentrations, with a total of three MRhos identified, including a high-acid-tolerance light-driven proton outward pump, HwBR, a chloride-insensitive chloride pump, HwHR, and a functionally unknown HwMR. Here, we showed that HwMR is the sole magnesium-sensitive MRho among all tested MRho proteins from Haloarchaea. We identified at least D84 as one of the key residues mediating such magnesium ion association in HwMR. Sequence analysis and molecular modeling suggested HwMR to have an extra H8 helix in the cytosolic region like those in signal-transduction-type MRho of deltarhodopsin-3 (dR-3) and Anabaena sensory rhodopsin (ASR). Further, HwMR showed a distinctly prolonged M-state formation under a high concentration of Mg2+. On the other hand, an H8 helix truncated mutant preserved photocycle kinetics like the wild type, but it led to missing M-state structure. Our findings clearly suggested not only that HwMR is a novel Mg2+-associated protein but that the association with both Mg2+ and the H8 domain stabilizes M-state formation in HwMR. We conclude that Mg2+ association and H8 are crucial in stabilizing HwMR M state, which is a well-known photoreceptor signaling state.

Dietary chlorella (Chlorella vulgaris) supplementation effectively improves body color, alleviates muscle inflammation and inhibits apoptosis in largemouth bass (Micropterus salmoides).

Muscle quality, antioxidant status, and inflammatory and apoptotic molecule expression were investigated in juvenile largemouth bass fed five levels of Chlorella for 60 days. The results showed that muscle quality can be improved by increasing the muscle crude protein content, muscle and skin brightness value (L*), redness value (a*) and yellowness value (b*) in Chlorella-supplemented diets without affecting the growth and muscle fiber development of fish. Chlorella supplementation did not cause oxidative stress in muscle, but optimal Chlorella administration alleviated the muscle inflammatory response by downregulating the nuclear factor κB (NF-κB)-mediated proinflammatory factors such as interleukin 1ß (IL-1ß) and interleukin 8 (IL-8). Moreover, anti-apoptotic effects were induced by upregulation of anti-apoptotic genes, such as b cell lymphoma-2 (bcl-2) and myeloid cell leukemia-1 (mcl-1), and downregulation of pro-apoptotic genes, including bcl2-associated x (bax) and caspase3. In conclusion, Chlorella improved muscle quality, alleviated muscle inflammation and resisted muscle apoptosis.

Effects of phytase supplementation of high-plant-protein diets on growth, phosphorus utilization, antioxidant, and digestion in red swamp crayfish (Procambarus clarkii).

Fish meal is increasingly being replaced by plant protein raw materials, meanwhile, it brings phytic acid, which combines with phosphorus to form phytate phosphorus and leads to a low utilization rate of phosphorus in shrimp. To solve this problem, this study investigated the effects of phytase supplementation on growth performance, phosphorus utilization, antioxidants, and digestion in red swamp crayfish (Procambarus clarkii). Crayfish (initial mean weight: 8.69 ± 0.15 g, N = 324) were randomly divided into six groups each with three replicates of 18 individuals each, and hand-fed for 8 weeks with one of six experimental diets (50 and 490 g kg-1 animal and plant protein raw material, respectively): negative control (NC; 11.0 g kg-1 phosphorus), positive control (PC; 15 g kg-1 NaH2PO4 added to NC; 14.7 g kg-1 phosphorus), and phytase supplementation diets (P1-P4: 0.1, 0.2, 0.4, and 0.6 g kg-1 phytase added to NC, respectively). The feeding trial was performed in a micro-flow water culture system. P2 showed a significantly higher weight gain rate (WGR), specific growth rate, protein efficiency ratio, and protein retention efficiency (PRE) but showed the lowest feed conversion ratio (FCR) than other groups. Broken-line regression analyses using WGR, FCR, and PRE as evaluation indices showed that the optimal dietary phytase supplementation level was 0.233, 0.244, and 0.303 g kg-1, respectively. P2 showed the highest crude protein content of whole crayfish and abdominal muscle, and phosphorus deposition rate, which was significantly higher than that in NC and PC. P3 showed the highest calcium and phosphorus contents in whole crayfish and phosphorus content in abdominal muscle, and calcium and inorganic phosphorus content in serum, which were significantly higher than those in NC. P3 showed significantly lowest serum alkaline phosphatase, alanine aminotransferase, aspartate transaminase activities, malondialdehyde content in hepatopancreas, and highest catalase activity, which were significantly lower and higher, respectively, than those in NC and PC. In summary, the addition of 0.2-0.4 g kg-1 phytase significantly improves the growth performance, feed utilization, digestive enzyme activity, and antioxidant of P. clarkii, which has a similar effect to the direct addition of NaH2PO4 at 15 g kg-1 to the feed.

Appropriate dietary phenylalanine improved growth, protein metabolism and lipid metabolism, and glycolysis in largemouth bass (Micropterus salmoides).

The purpose of this study was aimed to determine the appropriate level of dietary phenylalanine and explored the influences of phenylalanine on target rapamycin (TOR) signaling and glucose and lipid metabolism in largemouth bass. Six isonitrogenous/isoenergetic diets with graded phenylalanine levels (1.45% (control group), 1.69%, 1.98%, 2.21%, 2.48%, and 2.76%) were designed. Experimental feed was used to feed juvenile largemouth bass (initial body weight 19.5 ± 0.98 g) for 8 weeks. The final body weight, specific growth rate (SGR), feed efficiency ratio (FER), and weight gain (WG) reached their highest values in the 1.98% dietary phenylalanine group and then declined with increasing phenylalanine addition. No significant difference was found in the whole-body composition of largemouth bass between different dietary phenylalanine groups. Compared with the control group, 1.69% dietary phenylalanine significantly reduced the contents of plasma glucose (GLU) and total protein (TP), and total cholesterol (TC) contents increased significantly in the 1.98% dietary phenylalanine group (P < 0.05). The key gene expressions of TOR signaling pathway and lipid metabolism was significantly inhibited by 2.21% dietary phenylalanine (P < 0.05). The 1.98% dietary phenylalanine group showed significantly increased expression of genes related to insulin signaling pathway and factors involved in fatty acid synthesis (P < 0.05). Furthermore, 2.76% dietary phenylalanine group inhibited glucose metabolism by lowering the key gene expressions of glucose metabolism (P < 0.05). According to quadratic regression analyses based on the WG and FER, the appropriate level of dietary phenylalanine for largemouth bass were 2.00% and 2.02% of the diet (4.23% and 4.27% dietary protein), respectively, with a constant amount of tyrosine (1.33%). Hence, the total aromatic amino acid requirements were 3.33% and 3.35% of the diet (equivalent to 7.03% and 7.09% of the protein content), which may provide a theoretical basis for the development of largemouth bass feed formulas. Therefore, the growth and metabolism of largemouth bass could be promoted by controlling the content of phenylalanine in the diet, or the imbalance of phenylalanine can form a specific pathological model.

Operando Quantified Lithium Plating Determination Enabled by Dynamic Capacitance Measurement in Working Li-Ion Batteries.

The access to full performance of state-of-the-art Li-ion batteries (LIBs) is hindered by the mysterious lithium plating behavior. A rapid quantified lithium plating determination method compatible with actual working conditions is an urgent necessity for safe working LIBs. In this contribution, the relationship between electrical double layer (EDL) capacitance and electrochemical active surface area (ECSA) of graphite anodes during the Li-ion intercalation and Li plating processes is unveiled. We propose an operando lithium plating determination method based on the dynamic capacitance measurement (DCM) test. Reasonable selection of alternating current (AC) frequency protects the anodic responses from the interference of cathodic responses, which allows DCM to be applied in practical LIBs. The onset of lithium plating can be quantitatively traced, demonstrating the promise for real-time operando determination for lithium plating in a working battery.

Culture salinity modulates Nrf2 antioxidant signaling pathway and immune response of juvenile Genetically Improved Farmed Tilapia (GIFT) (Oreochromis niloticus) under different dietary protein levels.

This study aimed to evaluate that dietary protein levels and culture salinity levels affect the health status of juvenile genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Graded protein levels of six diets were prepared, ranging from 18.20% to 49.49% (dry basis), and were used in cultured GIFT at two salinity levels (0‰ and 8‰) for 8 weeks. The results suggested that appropriate protein levels reduced pro-inflammatory gene expressions in the intestine including interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels at two salinity levels (P < 0.05). 8‰ salinity significantly decreased the expression levels of IL-1ß, TNF-α and nuclear factor-kappa B (NF-κB) (P < 0.05). The anti-inflammatory factor interleukin 10 (IL-10) was significantly increased by 36.42% protein level (P < 0.05). Regarding antioxidant capacity, appropriate protein levels and 8‰ salinity significantly improved the antioxidant capacity of fish by regulating the activities of intestinal total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and the levels of glutathione (GSH) and malondialdehyde (MDA). Furthermore, appropriate protein levels and 8‰ salinity also significantly enhanced the antioxidant gene expressions associated with the Nrf2/keap1 signaling pathway by regulating the expression levels of heme oxygenase-1 (HO-1), GPx, catalase (CAT) and superoxide dismutase (SOD). According to GPx activities and the mRNA levels of IL-10, the optimum dietary protein levels for GIFT juveniles were 31.12%-32.18% (0‰) and 34.25-35.38% (8‰) based on second-degree polynomial regression analysis. The present study found that appropriate protein levels and 8‰ culture salinity are critical in maintaining the health of GIFT juveniles by improving antioxidant and immune capacity.

The Boundary of Lithium Plating in Graphite Electrode for Safe Lithium-Ion Batteries.

Uncontrolled Li plating in graphite electrodes endangers battery life and safety, driving tremendous efforts aiming to eliminate Li plating. Herein we systematically investigate the boundary of Li plating in graphite electrode for safe lithium-ion batteries. The cell exhibits superior safety performance than that with Li dendrites by defining the endurable amount of uniform Li plating in graphite anode. The presence of "dead Li" can be eliminated owing to the uniform distribution of Li plating, and the average Coulombic efficiency for deposited Li during reversible plating/stripping process is decoupled as high as about 99.5 %. Attributing to the limited Li plating with superior Coulombic efficiency, the LiNi0.5 Mn0.3 Co0.2 O2 | graphite cell achieves a high capacity retention of 80.2 % over 500 cycles. This work sheds a different light on further improving the fast-charging capability, low-temperature performance, and energy density of practical lithium-ion batteries.

A Diffusion--Reaction Competition Mechanism to Tailor Lithium Deposition for Lithium-Metal Batteries.

Lithium metal is recognized as one of the most promising anode materials owing to its ultrahigh theoretical specific capacity and low electrochemical potential. Nonetheless, dendritic Li growth has dramatically hindered the practical applications of Li metal anodes. Realizing spherical Li deposition is an effective approach to avoid Li dendrite growth, but the mechanism of spherical deposition is unknown. Herein, a diffusion-reaction competition mechanism is proposed to reveal the rationale of different Li deposition morphologies. By controlling the rate-determining step (diffusion or reaction) of Li deposition, various Li deposition scenarios are realized, in which the diffusion-controlled process tends to lead to dendritic Li deposition while the reaction-controlled process leads to spherical Li deposition. This study sheds fresh light on the dendrite-free Li metal anode and guides to achieve safe batteries to benefit future wireless and fossil-fuel-free world.

Schiff Base Proton Acceptor Assists Photoisomerization of Retinal Chromophores in Bacteriorhodopsin.

In this study, we investigated the ultrafast dynamics of bacteriorhodopsins (BRs) from Haloquadratum walsbyi (HwBR) and Haloarcula marismortui (HmBRI and HmBRII). First, the ultrafast dynamics were studied for three HwBR samples: wild-type, D93N mutation, and D104N mutation. The residues of the D93 and D104 mutants correspond to the control by the Schiff base proton acceptor and donor of the proton translocation subchannels. Measurements indicated that the negative charge from the Schiff base proton acceptor residue D93 interacts with the ultrafast and substantial change of the electrostatic potential associated with chromophore isomerization. By contrast, the Schiff base proton donor assists the restructuring of the chromophore cavity hydrogen-bond network during the thermalization of the vibrational hot state. Second, the ultrafast dynamics of the wild-types of HwBR, HmBRI, and HmBRII were compared. Measurements demonstrated that the hydrogen-bond network in the extracellular region in HwBR and HmBRII slows the photoisomerization of retinal chromophores, and the negatively charged helices on the cytoplasmic side of HwBR and HmBRII accelerate the thermalization of the vibrational hot state of retinal chromophores. The similarity of the correlation spectra of the wild-type HmBRI and D104N mutant of HwBR indicates that inactivation of the Schiff base proton donor induces a positive charge on the helices of the cytoplasmic side.

Characterization of the anti-CD22 targeted therapy, moxetumomab pasudotox, for B-cell precursor acute lymphoblastic leukemia.

Moxetumomab pasudotox is a second-generation recombinant immunotoxin against CD22 on B-cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy-refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient-derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD-scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient-derived xenograft models.

Lithiophilic Sites in Doped Graphene Guide Uniform Lithium Nucleation for Dendrite-Free Lithium Metal Anodes.

Lithium (Li) metal is the most promising electrode for next-generation rechargeable batteries. However, the challenges induced by Li dendrites on a working Li metal anode hinder the practical applications of Li metal batteries. Herein, nitrogen (N) doped graphene was adopted as the Li plating matrix to regulate Li metal nucleation and suppress dendrite growth. The N-containing functional groups, such as pyridinic and pyrrolic nitrogen in the N-doped graphene, are lithiophilic, which guide the metallic Li nucleation causing the metal to distribute uniformly on the anode surface. As a result, the N-doped graphene modified Li metal anode exhibits a dendrite-free morphology during repeated Li plating and demonstrates a high Coulombic efficiency of 98 % for near 200 cycles.

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis.

Halorhodopsin (HR) is a seven-transmembrane retinylidene protein from haloarchaea that is commonly known to function as a light-driven inward chloride pump. However, previous studies have indicated that despite the general characteristics that most HRs share, HRs from distinct species differ in many aspects. We present indium-tin-oxide-based photocurrent measurements that reveal a light-induced signal generated by proton release that is observed solely in NpHR via purified protein-based assays, demonstrating that indeed HRs are not all identical. We conducted mutagenesis studies on several conserved residues that are considered critical for chloride stability among HRs. Intriguingly, the photocurrent signals were eliminated after specific point mutations. We propose an NpHR light-driven, cytoplasmic-side proton circulation model to explain the unique light-induced photocurrent recorded in NpHR. Notably, the photocurrent and various photocycle intermediates were recorded simultaneously. This approach provides a high-resolution method for further investigations of the proton-assisted chloride translocation mechanism.

Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors.

The orphan GluD2 receptor belongs to the ionotropic glutamate receptor family but does not bind glutamate. Ligand-gated GluD2 currents have never been evidenced, and whether GluD2 operates as an ion channel has been a long-standing question. Here, we show that GluD2 gating is triggered by type 1 metabotropic glutamate receptors, both in a heterologous expression system and in Purkinje cells. Thus, GluD2 is not only an adhesion molecule at synapses but also works as a channel. This gating mechanism reveals new properties of glutamate receptors that emerge from their interaction and opens unexpected perspectives regarding synaptic transmission and plasticity.

Development of an Evaluation System for the Prophylactic Use of Antimicrobial Drugs in the Perioperative Period of Class I Surgical Incisions in Neurosurgery.

BACKGROUND: This study aimed to establish a precise preoperative high-risk factor scoring system and algorithm for antibiotic prophylaxis decision-making, provide guidance for the judicious use of AMP, refine interventions, and ensure the appropriate application of AMP for class I incisions in neurosurgery. METHODS: According to PRISMA guidelines, literature searches, study selection, methodology development, and quality appraisal were performed. The quality of evidence across the study population was assessed using the Newcastle-Ottawa Scale. A two-round Delphi expert consultation method involved 15 experts from leading tertiary hospitals in China. Establishing an algorithm of SOPs for perioperative antimicrobial prophylaxis in Class I neurosurgical incisions. RESULTS: Thirteen studies, encompassing 11,936 patients undergoing clean neurosurgical procedures, were included. 791 patients experienced SSI, resulting in an average incidence of 6.62%. Identified risk factors significantly associated with an increased incidence of postoperative SSI (P < 0.05) included emergency surgery, preoperative hospitalization ≥7 days, intraoperative blood loss ≥300 mL, operation time ≥4 hours, diabetes mellitus, cerebrospinal fluid leakage, and repeat surgery. Sensitivity analysis demonstrated robust results for emergency surgery, intraoperative blood loss ≥300 mL, operation time ≥4 hours, cerebrospinal fluid leakage, and repeat surgery. Established a risk assessment system for Class I neurosurgical incisions by the Delphi method. Additionally, we have formulated an algorithm of SOPs for perioperative antimicrobial prophylaxis in Class I neurosurgical incisions. CONCLUSIONS: The established index for AMP utilization and SOPs in the preoperative period of class I neurosurgical incisions proves valuable, contributing to improved patient outcomes in neurosurgical procedures.

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.