Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2

Nucleosomes are dynamic entities with wide‐ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo‐EM structures of nucleosome core particles containing human H2A.B (H2A.B‐NCP) at atomic resolution, identifying large‐scale structural rearrangements in the histone octamer in H2A.B‐NCP. H2A.B‐NCP compacts approximately 103 bp of DNA wrapping around the core histones in approximately 1.2 left‐handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B‐specific residues, including a ROF (“regulating‐octamer‐folding”) sequence of six consecutive residues, are responsible for loosening of H2A.B‐NCPs. Unlike H2A.B‐NCP, the H2A.Z.2.2‐containing nucleosome (Z.2.2‐NCP) adopts a less‐extended structure and compacts around 125 bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2‐specific ROF in both H2A.Z.2.2‐NCP opening and SWR1‐dependent histone replacement. Taken together, these first high‐resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.     

Neuronal α‐amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology

Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha‐amylase (α‐amylase), a glycogen degradation enzyme, located within synaptic‐like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α‐amylase perform better on the episodic memory test. We reported that neuronal α‐amylase was absent in patients with Alzheimer''s disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α‐amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α‐amylase concentrations in oligomer amyloid beta 42 (Aβ₄₂) stimulated SH‐SY5Y cells and neurons derived from human‐induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α‐amylase production and activity, induced by siRNA and α‐amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH‐SY5Y cells. Both oligomer Aβ₄₂ stimulated SH‐SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α‐amylase within synapses of isolated primary neurons and show that inhibition of α‐amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α‐amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α‐amylase, which can be induced by Aβ pathology, may in part underlie the disrupted memory formation seen in AD patients.     

Cryo‐EM structures of pentameric autoinducer‐2 exporter from Escherichia coli reveal its transport mechanism

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer‐2 (AI‐2), a universal molecule for both intra‐ and inter‐species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI‐2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI‐2 exporter superfamily, has been shown to export AI‐2. Here, we report the cryogenic electron microscopic structures of two AI‐2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI‐2 exporter exists as a homo‐pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI‐2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator‐type transport mechanism.     

Psychotherapies for depression: a network meta‐analysis covering efficacy,acceptability and long‐term outcomes of all main treatment types

The effects of psychotherapies for depression have been examined in several hundreds of randomized trials, but no recent network meta‐analysis (NMA) has integrated the results of these studies. We conducted an NMA of trials comparing cognitive behavioural, interpersonal, psychodynamic, problem‐solving, behavioural activation, life‐review and “third wave” therapies and non‐directive supportive counseling with each other and with care‐as‐usual, waiting list and pill placebo control conditions. Response (50% reduction in symptoms) was the primary outcome, but we also assessed remission, standardized mean difference, and acceptability (all‐cause dropout rate). Random‐effects pairwise and network meta‐analyses were conducted on 331 randomized trials with 34,285 patients. All therapies were more efficacious than care‐as‐usual and waiting list control conditions, and all therapies – except non‐directive supportive counseling and psychodynamic therapy – were more efficacious than pill placebo. Standardized mean differences compared with care‐as‐usual ranged from –0.81 for life‐review therapy to –0.32 for non‐directive supportive counseling. Individual psychotherapies did not differ significantly from each other, with the only exception of non‐directive supportive counseling, which was less efficacious than all other therapies. The results were similar when only studies with low risk of bias were included. Most therapies still had significant effects at 12‐month follow‐up compared to care‐as‐usual, and problem‐solving therapy was found to have a somewhat higher long‐term efficacy than some other therapies. No consistent differences in acceptability were found. Our conclusion is that the most important types of psychotherapy are efficacious and acceptable in the acute treatment of adult depression, with few significant differences between them. Patient preference and availability of each treatment type may play a larger role in the choice between types of psychotherapy, although it is possible that a more detailed characterization of patients with a diagnosis of depression may lead to a more precise matching between individual patients and individual psychotherapies.     

Optogenetic actuator – ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics

Combining single‐cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single‐cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK‐RAF and the ERK‐RSK2‐SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2‐mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2‐dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2‐mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.     

Aromaticity at position 39 in α‐synuclein: A modulator of amyloid fibril assembly and membrane‐bound conformations

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid‐binding features of αS through the design of site‐directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane‐bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death.Brief statement outlining significanceModulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane‐bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.     

Cryo‐EM structures of perforin‐2 in isolation and assembled on a membrane suggest a mechanism for pore formation

Perforin‐2 (PFN2, MPEG1) is a key pore‐forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane‐bound pre‐pore complex that converts to a pore‐forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo‐electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre‐pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre‐assembled complete pre‐pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre‐pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β‐hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre‐pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.     

Phospho‐regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.     

Cyclin D3 restricts SARS‐CoV‐2 envelope incorporation into virions and interferes with viral spread

The COVID‐19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) presents a great threat to human health. The interplay between the virus and host plays a crucial role in successful virus replication and transmission. Understanding host–virus interactions are essential for the development of new COVID‐19 treatment strategies. Here, we show that SARS‐CoV‐2 infection triggers redistribution of cyclin D1 and cyclin D3 from the nucleus to the cytoplasm, followed by proteasomal degradation. No changes to other cyclins or cyclin‐dependent kinases were observed. Further, cyclin D depletion was independent of SARS‐CoV‐2‐mediated cell cycle arrest in the early S phase or S/G2/M phase. Cyclin D3 knockdown by small‐interfering RNA specifically enhanced progeny virus titres in supernatants. Finally, cyclin D3 co‐immunoprecipitated with SARS‐CoV‐2 envelope (E) and membrane (M) proteins. We propose that cyclin D3 impairs the efficient incorporation of envelope protein into virions during assembly and is depleted during SARS‐CoV‐2 infection to restore efficient assembly and release of newly produced virions.     

Influenza A viruses limit NLRP3‐NEK7‐complex formation and pyroptosis in human macrophages

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro‐inflammatory cytokines. In humans, it depends on caspase 1/4‐activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1‐F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1‐F2‐deficient mutant of a contemporary IAV resulted in higher levels of caspase‐1 activation, cleavage of gasdermin D, and release of LDH and IL‐1β. Mechanistically, PB1‐F2 limits transition of NLRP3 from its auto‐repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.     

Mammalian adipogenesis regulator (Areg) cells use retinoic acid signalling to be non‐ and anti‐adipogenic in age‐dependent manner

Adipose stem and precursor cells (ASPCs) give rise to adipocytes and determine the composition and plasticity of adipose tissue. Recently, several studies have demonstrated that ASPCs partition into at least three distinct cell subpopulations, including the enigmatic CD142⁺ cells. An outstanding challenge is to functionally characterise this population, as discrepant properties, from adipogenic to non‐ and anti‐adipogenic, have been reported for these cells. To resolve these phenotypic ambiguities, we characterised mammalian subcutaneous CD142⁺ ASPCs across various experimental conditions, demonstrating that CD142⁺ ASPCs exhibit high molecular and phenotypic robustness. Specifically, we find these cells to be firmly non‐ and anti‐adipogenic both in vitro and in vivo, with their inhibitory signals also impacting adipogenic human cells. However, these CD142⁺ ASPC‐specific properties exhibit surprising temporal phenotypic alterations, and emerge only in an age‐dependent manner. Finally, using multi‐omic and functional assays, we show that the inhibitory nature of these adipogenesis‐regulatory CD142⁺ ASPCs (Aregs) is driven by specifically expressed secretory factors that cooperate with the retinoic acid signalling pathway to transform the adipogenic state of CD142⁻ ASPCs into a non‐adipogenic, Areg‐like state.     

Circular RNA circEIF4G2 aggravates renal fibrosis in diabetic nephropathy by sponging miR‐218

Circular RNAs play essential roles in the development of various human diseases. However, how circRNAs are involved in diabetic nephropathy (DN) are not fully understood. Our study aimed to investigate the effects of circRNA circEIF4G2 on DN. Experiments were performed in the db/db mouse model of type 2 diabetes and NRK‐52E cells. We found that circEIF4G2 was significantly up‐regulated in the kidneys of db/db mice and NRK‐52E cells stimulated by high glucose. circEIF4G2 knockdown inhibited the expressions of TGF‐β1, Collagen I and Fibronectin in high glucose‐stimulated NRK‐52E cells, which could be rescued by miR‐218 inhibitor. Knockdown of SERBP1 reduced the expression of TGF‐β1, Collagen I and Fibronectin in HG‐stimulated NRK‐52E cells. In summary, our findings suggested that circEIF4G2 promotes renal tubular epithelial cell fibrosis via the miR‐218/SERBP1 pathway, presenting a novel insight for DN treatment.     

Neuropeptide Y regulates cholesterol uptake and efflux in macrophages and promotes foam cell formation

The dysregulation of lipid metabolic pathways (cholesterol uptake and efflux) in macrophages results in the formation of lipid‐dense macrophages, named foam cells, that participate in plaque formation. NPY binding to NPY receptors in macrophages can modulate cell functions and affect the process of atherosclerotic plaques. The present study aimed to determine whether NPY affects the formation of macrophage‐derived foam cells and its underlying mechanisms in macrophages. THP‐1‐derived macrophages were incubated with oxidized low‐density lipoprotein (ox‐LDL) and treated with different concentrations of NPY. We analysed the relative levels of proteins related to cholesterol uptake and efflux. We found that NPY effectively increased cholesterol uptake and intracellular cholesterol content via the Y1 and Y5 receptors, and this effect was blocked by Y1 and Y5 antagonists. Mechanistically, NPY enhanced the expression of SRA and CD36 via the PKC/PPARγ pathways, promoting macrophage cholesterol uptake. Moreover, NPY significantly decreased cholesterol efflux to the extracellular cholesterol acceptors ApoA1 and HDL in macrophages. NPY mediated decreases in ABCA1, ABCG1 and SR‐BI expression through the inhibition of the JAK/STAT3 pathways. Our results suggest that NPY binding to the Y1 and Y5 receptors enhances foam cell formation by regulating cholesterol uptake and efflux in macrophages.     

Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1‐CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1‐CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1‐CTDNEP1 downregulation also restores nuclear pore membrane fusion in post‐mitotic dTorsin^KO fat body cells. However, dTorsin‐associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel‐like structures in place of mature nuclear pores. We conclude that the NEP1R1‐CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid‐dependent and lipid‐independent mechanisms, explaining some of the pleiotropic effects of Torsins.     

Cryptic association of B7‐2 molecules and its implication for clustering

T‐cell co‐stimulation through CD28/CTLA4:B7‐1/B7‐2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA‐approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7‐2 on the cell surface. Here, we describe the structure of the IgV domain of B7‐2 and its cryptic association into 1D arrays that appear to represent the pre‐signaling state of B7‐2 on the cell membrane. Super‐resolution microscopy experiments on heterologous cells expressing B7‐2 and B7‐1 suggest, B7‐2 form relatively elongated and larger clusters compared to B7‐1. The sequence and structural comparison of other B7 family members, B7‐1:CTLA4 and B7‐2:CTLA‐4 complex structures, support our view that the observed B7‐2 1D zipper array is physiologically important. This observed 1D zipper‐like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.     

Architecture of the active post‐translational Sec translocon

In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel (PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex.