Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.

Discovery of High Affinity Cyclic Peptide Ligands for Human ACE2 with SARS-CoV-2 Entry Inhibitory Activity.

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.

mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome.

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.

A view from mindreading on fast-and-slow thinking.

De Neys's incisive critique of empirical and theoretical research on the exclusivity feature underscores the depth of the challenge of explaining the interplay of fast and slow processes. We argue that a closer look at research on mindreading reveals abundant evidence for the exclusivity feature - as well as methodological and theoretical perspectives that could inform research on fast and slow thinking.

Mindreading by body: incorporating mediolateral balance and mouse-tracking measures to examine the motor basis of adults' false-belief tracking.

The role played by motor representations in tracking others' belief-based actions remains unclear. In experiment 1, the dynamics of adults' anticipatory mediolateral motor activity (leftwards-rightwards leaning on a balance board) as well as hand trajectories were measured as they attempted to help an agent who had a true or false belief about an object's location. Participants' leaning was influenced by the agent's belief about the target's location when the agent was free to act but not when she was motorically constrained. However, the hand trajectories participants produced to provide a response were not modulated by the other person's beliefs. Therefore, we designed a simplified second experiment in which participants were instructed to click as fast as possible on the location of a target object. In experiment 2, mouse-movements deviated from an ideal direct path to the object location, with trajectories that were influenced by the location in which the agent falsely believed the object to be located. These experiments highlight that information about an agent's false-belief can be mapped onto the motor system of a passive observer, and that there are situations in which the motor system plays an important role in accurate belief-tracking.

Cross-linking mass spectrometry discovers, evaluates, and corroborates structures and protein-protein interactions in the human cell.

Significant recent advances in structural biology, particularly in the field of cryoelectron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability, or-in the case of complexes-simply not having yet been analyzed. Here, we demonstrate the power of using cross-linking mass spectrometry (XL-MS) for the high-throughput experimental assessment of the structures of proteins and protein complexes. This included those produced by high-resolution but in vitro experimental data, as well as in silico predictions based on amino acid sequence alone. We present the largest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein-protein interactions. We show that models of proteins and their complexes predicted by AlphaFold2, and inspired and corroborated by the XL-MS data, offer opportunities to deeply mine the structural proteome and interactome and reveal mechanisms underlying protein structure and function.

Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional programs.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Pathogenic human variant that dislocates GATA2 zinc fingers disrupts hematopoietic gene expression and signaling networks.

Although certain human genetic variants are conspicuously loss of function, decoding the impact of many variants is challenging. Previously, we described a patient with leukemia predisposition syndrome (GATA2 deficiency) with a germline GATA2 variant that inserts 9 amino acids between the 2 zinc fingers (9aa-Ins). Here, we conducted mechanistic analyses using genomic technologies and a genetic rescue system with Gata2 enhancer-mutant hematopoietic progenitor cells to compare how GATA2 and 9aa-Ins function genome-wide. Despite nuclear localization, 9aa-Ins was severely defective in occupying and remodeling chromatin and regulating transcription. Variation of the inter-zinc finger spacer length revealed that insertions were more deleterious to activation than repression. GATA2 deficiency generated a lineage-diverting gene expression program and a hematopoiesis-disrupting signaling network in progenitors with reduced granulocyte-macrophage colony-stimulating factor (GM-CSF) and elevated IL-6 signaling. As insufficient GM-CSF signaling caused pulmonary alveolar proteinosis and excessive IL-6 signaling promoted bone marrow failure and GATA2 deficiency patient phenotypes, these results provide insight into mechanisms underlying GATA2-linked pathologies.

A NuRD for all seasons.

The nucleosome-remodeling and deacetylase (NuRD) complex is an essential transcriptional regulator in all complex animals. All seven core subunits of the complex exist as multiple paralogs, raising the question of whether the complex might utilize paralog switching to achieve cell type-specific functions. We examine the evidence for this idea, making use of published quantitative proteomic data to dissect NuRD composition in 20 different tissues, as well as a large-scale CRISPR knockout screen carried out in >1000 human cancer cell lines. These data, together with recent reports, provide strong support for the idea that distinct permutations of the NuRD complex with tailored functions might regulate tissue-specific gene expression programs.

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families.

CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by ~150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

The psychology of natural history.

Natural history observations are an integral part of ecology and evolution. However, they can be underappreciated because they operate independent of the scientific method. Here, we illustrate that the science of natural history has its own methodology based on a well-known psychological paradigm that describes how the human mind learns.

Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination.

The nucleosome remodeling and deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information describing the complete NuRD complex is available and a detailed understanding of its mechanism is therefore lacking. Drawing on information from SEC-MALLS, DIA-MS, XLMS, negative-stain EM, X-ray crystallography, NMR spectroscopy, secondary structure predictions, and homology models, we applied Bayesian integrative structure determination to investigate the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4, MTA1N -HDAC1-MBD3GATAD2CC , and MTA1-HDAC1-RBBP4-MBD3-GATAD2A [nucleosome deacetylase (NuDe)]. The integrative structures were corroborated by examining independent crosslinks, cryo-EM maps, biochemical assays, known cancer-associated mutations, and structure predictions from AlphaFold. The robustness of the models was assessed by jack-knifing. Localization of the full-length MBD3, which connects the deacetylase and chromatin remodeling modules in NuRD, has not previously been possible; our models indicate two different locations for MBD3, suggesting a mechanism by which MBD3 in the presence of GATAD2A asymmetrically bridges the two modules in NuRD. Further, our models uncovered three previously unrecognized subunit interfaces in NuDe: HDAC1C -MTA1BAH , MTA1BAH -MBD3MBD , and HDAC160-100 -MBD3MBD . Our approach also allowed us to localize regions of unknown structure, such as HDAC1C and MBD3IDR , thereby resulting in the most complete and robustly cross-validated structural characterization of these NuRD sub-complexes so far.

Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability.

The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

Unique protein interaction networks define the chromatin remodelling module of the NuRD complex.

The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1 and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodelling module of the nucleosome remodelling and deacetylase (NuRD) complex. To date, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential for defining underlying mechanisms of gene regulation. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS), we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodelling activity into both ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner. We define a short aggregation-prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the nuclear receptor co-repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

Retrieval-induced forgetting for autobiographical memories beyond recall rates: A developmental study.

We investigated whether selective discussion of autobiographical memory narratives would impact the quality of young people's recall of their nondiscussed memory narratives. Children (ages 8-9 years, n = 65) and adolescents (ages 13-15 years, n = 58) completed an adapted version of the retrieval-induced forgetting (RIF) paradigm for self-generated positively and negatively valenced autobiographical memories. Overall, 54.5% of the sample were female and 63.4% were of European ethnicity (11.4% Pacific Peoples, 8.1% Middle Eastern/Latin American/African, 7.3% Maori, 7.3% Asian, 2.4% Other). We defined narrative qualities as narrative coherence (Reese et al., 2011) and episodic and nonepisodic information (Addis et al., 2008). In light of developmental findings in other domains of autobiographical memory research (e.g., Reese et al., 2011; Willoughby et al., 2012), we hypothesized that selective discussion would result in RIF for children's, but not adolescents', narrative coherence and episodic detail, and that RIF would not occur for nonepisodic details for either children or adolescents. Findings for narrative coherence and nonepisodic detail indicated support for our hypotheses. Findings for episodic detail were in partial support of our hypothesis; RIF for episodic detail was found for both children and adolescents. Our findings not only demonstrate the importance of investigating the wider effects of RIF but also uncovered developmental differences previously overlooked in the field. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilising immunity against SARS-CoV-2 infection.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation.

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

The characterization of protein interactions - what, how and how much?

Protein interactions underlie most molecular events in biology. Many methods have been developed to identify protein partners, to measure the affinity with which these biomolecules interact and to characterise the structures of the complexes. Each approach has its own advantages and limitations, and it can be difficult for the newcomer to determine which methodology would best suit their system. This review provides an overview of many of the techniques most widely used to identify protein partners, assess stoichiometry and binding affinity, and determine low-resolution models for complexes. Key methods covered include: yeast two-hybrid analysis, affinity purification mass spectrometry and proximity labelling to identify partners; size-exclusion chromatography, scattering methods, native mass spectrometry and analytical ultracentrifugation to estimate stoichiometry; isothermal titration calorimetry, biosensors and fluorometric methods (including microscale thermophoresis, anisotropy/polarisation, resonance energy transfer, AlphaScreen, and differential scanning fluorimetry) to measure binding affinity; and crosslinking and hydrogen-deuterium exchange mass spectrometry to probe the structure of complexes.

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein Using mRNA Display.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality, and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein, and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (K D) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect the spike protein in solution by ELISA, and the cocrystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a ß-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD, and the ligands discovered in this work may find future use as reagents for diagnostic applications.

Association Between Diabetes Mellitus and Carpal Tunnel Syndrome: Results From the United States National Ambulatory Medical Care Survey.

Background Carpal tunnel syndrome (CTS) is the most common compression neuropathy in the upper limb. While various risk factors have been linked to CTS, the role of diabetes mellitus (DM) in the development of CTS remains unclear. Previous studies have failed to consistently demonstrate a clear association between DM and CTS due to variations based on the geographic setting and differences in the study design. The objective of this study was to assess if there is an association between DM and CTS using population-based data from the United States. Methodology We used data from patients ≥18 years old who contributed to the National Ambulatory Medical Care Survey between 2006 and 2015. The outcome was CTS identified by the International Classification of Diseases-9-Clinical Modification codes (354.0 and 354.1), and the main independent variable was physician-reported diabetes status. Multivariable logistic regression was used to adjust for confounding variables. Odds ratios (ORs) and 95% confidence intervals (CIs) were reported. Stata v15 was used for all analyses. Results Among the patients included in this study (n = 322,092), 13.5% were reported to have diabetes while 0.55% reported CTS. The unadjusted odds of having CTS among patients with diabetes was 0.92 (95% CI: 0.74-1.14; p = 0.447). After adjusting for confounding variables, the association remained not statistically significant (adjusted odds ratio [aOR]: 0.84; 95% CI: 0.65-1.09; p = 0.203). Other variables independently associated with CTS included age 50-59 (aOR: 1.91; 95% CI: 1.49-2.45; p < 0.001), female gender (aOR: 1.31; 95% CI: 1.09-1.58; p < 0.004), and current tobacco users (aOR: 1.32; 95% CI: 1.07-1.63; p < 0.01). Conclusions No association was found between DM and CTS in adult ambulatory patients in the United States, but results should be considered in light of potential outcome misclassification.