Estrogen receptor alpha (ERα) signaling pathway is essential for ERα-positive breast cancer progression and endocrine therapy resistance. Bromodomain PHD Finger Transcription Factor (BPTF) associated protein of 18kDa (BAP18) has been recognized as a crucial H3K4me3 reader. However, the whole genomic occupation of BAP18 and its biological function in breast cancer is still elusive. Here, we found that higher expression of BAP18 in ERα-positive breast cancer is positively correlated with poor prognosis. ChIP-seq analysis further demonstrated that the half estrogen response elements (EREs) and the CCCTC binding factor (CTCF) binding sites are the significant enrichment sites found in estrogen-induced BAP18 binding sites. Also, we provide the evidence to demonstrate that BAP18 as a novel co-activator of ERα is required for the recruitment of COMPASS-like core subunits to the cis-regulatory element of ERα target genes in breast cancer cells. BAP18 is recruited to the promoter regions of estrogen-induced genes, accompanied with the enrichment of the lysine 4-trimethylated histone H3 tail (H3K4me3) in the presence of E2. Furthermore, BAP18 promotes cell growth and associates the sensitivity of antiestrogen in ERα-positive breast cancer. Our data suggest that BAP18 facilitates the association between ERα and COMPASS-like core subunits, which might be an essential epigenetic therapeutic target for breast cancer.

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.

Hypoxia-inducible factor (HIF) controls an extensive range of adaptive responses to hypoxia. To better understand this transcriptional cascade we performed genome-wide chromatin immunoprecipitation using antibodies to two major HIF-alpha subunits, and correlated the results with genome-wide transcript profiling. Within a tiled promoter array we identified 546 and 143 sequences that bound, respectively, to HIF-1alpha or HIF-2alpha at high stringency. Analysis of these sequences confirmed an identical core binding motif for HIF-1alpha and HIF-2alpha (RCGTG) but demonstrated that binding to this motif was highly selective, with binding enriched at distinct regions both upstream and downstream of the transcriptional start. Comparison of HIF-promoter binding data with bidirectional HIF-dependent changes in transcript expression indicated that whereas a substantial proportion of positive responses (>20% across all significantly regulated genes) are direct, HIF-dependent gene suppression is almost entirely indirect. Comparison of HIF-1alpha- versus HIF-2alpha-binding sites revealed that whereas some loci bound HIF-1alpha in isolation, many bound both isoforms with similar affinity. Despite high-affinity binding to multiple promoters, HIF-2alpha contributed to few, if any, of the transcriptional responses to acute hypoxia at these loci. Given emerging evidence for biologically distinct functions of HIF-1alpha versus HIF-2alpha understanding the mechanisms restricting HIF-2alpha activity will be of interest.

Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1-452, and is phosphorylated at Ser435 and Thr440. Residues 1-429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be a putative Gα-binding surface. Normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1-4, 5-6, and 7-9. 2D 1H-15N-TROSY spectra of [2H,15N]-labeled Gαi1 in the presence of R452 reveals chemical shift perturbations of the C terminus and Gαi1 residues involved in nucleotide binding.

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have arisen that exhibit increased viral transmissibility and partial evasion of immunity induced by natural infection and vaccination. To address the specific antibody targets that were affected by recent viral variants, we generated 43 monoclonal antibodies (mAbs) from 10 convalescent donors that bound three distinct domains of the SARS-CoV-2 spike. Viral variants harboring mutations at K417, E484, and N501 could escape most of the highly potent antibodies against the receptor binding domain (RBD). Despite this, we identified 12 neutralizing mAbs against three distinct regions of the spike protein that neutralize SARS-CoV-2 and variants of concern (VOCs), including B.1.1.7 (alpha), P.1 (gamma), and B.1.617.2 (delta). Notably, antibodies targeting distinct epitopes could neutralize discrete variants, suggesting that different variants may have evolved to disrupt the binding of particular neutralizing antibody classes. These results underscore that humans exposed to the first pandemic wave of prototype SARS-CoV-2 possess neutralizing antibodies against current variants and that it is critical to induce antibodies targeting multiple distinct epitopes of the spike that can neutralize emerging variants of concern. IMPORTANCE We describe the binding and neutralization properties of a new set of human monoclonal antibodies derived from memory B cells of 10 coronavirus disease 2019 (COVID-19) convalescent donors in the first pandemic wave of prototype SARS-CoV-2. There were 12 antibodies targeting distinct epitopes on spike, including two sites on the RBD and one on the N-terminal domain (NTD), that displayed cross-neutralization of VOCs, for which distinct antibody targets could neutralize discrete variants. This work underlines that natural infection by SARS-CoV-2 induces effective cross-neutralization against only some VOCs and supports the need for COVID-19 vaccination for robust induction of neutralizing antibodies targeting multiple epitopes of the spike protein to combat the current SARS-CoV-2 VOCs and any others that might emerge in the future.

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.

In Drosophila, the piRNAs that guide germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. We show that Rhino promotes cluster transcript association with UAP56 and the THO complex, forming RNA-protein assemblies that are unique to piRNA precursors. UAP56 and THO are ubiquitous RNA-processing factors, and null alleles of uap56 and the THO subunit gene tho2 are lethal. However, uap56sz15 and mutations in the THO subunit genes thoc5 and thoc7 are viable but sterile and disrupt piRNA biogenesis. The uap56sz15 allele reduces UAP56 binding to THO, and the thoc5 and thoc7 mutations disrupt interactions among the remaining THO subunits and UAP56 binding to the core THO subunit Hpr1. These mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino at cluster heterochromatin.

To understand the function of multisubunit complexes, it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here, we demonstrate that the core modules of ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription co-activator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, a SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histone proteins. In contrast, ATAC complex subunits cannot be detected in the cytoplasm of mammalian cells. However, an endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related co-activators, ATAC and SAGA, assemble using co-translational pathways, but their subcellular localization, cytoplasmic abundance, and functions are distinct.

Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.

HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. Here, we characterize PCI domain-containing 2 (PCID2) protein, a subunit of the transcription and export complex 2 (TREX2) complex, to enforce transcriptional repression and post-transcriptional blocks to HIV-1 gene expression during latency. PCID2 bound the latent HIV-1 LTR (long terminal repeat) and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and latency reversal. Immunoprecipitation coupled to mass spectrometry identified PCID2-interacting proteins to include negative viral RNA (vRNA) splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA in cell lines and primary cells obtained from PWH. MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits, also inhibit transcription initiation and vRNA alternative splicing during latency. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.

Glutaminase is a multifunctional enzyme encoded by gene Gls involved in energy metabolism, ammonia trafficking and regeneration of neurotransmitter glutamate. To address the proteomic basis for the neurophenotypes of glutaminase-deficient mice, brain proteins from late gestation wild type, Gls+/- and Gls-/- male mice were subjected to two-dimensional gel electrophoresis, with subsequent identification by mass spectrometry using nano-LC-ESI-MS/MS. Protein spots that showed differential genotypic variation were quantified by immunoblotting. Differentially expressed proteins unambiguously identified by MS/MS included neurocalcin delta, retinol binding protein-1, reticulocalbin-3, cytoskeleton proteins fascin and tropomyosin alpha-4-chain, dihydropyrimidinase-related protein-5, apolipoprotein IV and proteins from protein metabolism proteasome subunits alpha type 2, type 7, heterogeneous nuclear ribonucleoprotein C1/C2 and H, voltage-gated anion-selective channel proteins 1 and 2, ATP synthase subunit β and transitional endoplasmic reticulum ATPase. An interaction network determined by Ingenuity Pathway Analysis revealed a link between glutaminase and calcium, Akt and retinol signaling, cytoskeletal elements, ATPases, ion channels, protein synthesis and the proteasome system, intermediary, nucleic acid and lipid metabolism, huntingtin, guidance cues, transforming growth factor beta-1 and hepatocyte nuclear factor 4-alpha. The network identified involves (a) cellular assembly and organization and (b) cell signaling and cell cycle, suggesting that Gls is crucial for neuronal maturation.

Increased pro-inflammatory signaling is a hallmark of metabolic dysfunction in obesity and diabetes. Although both inflammatory and energy substrate handling processes represent critical layers of metabolic control, their molecular integration sites remain largely unknown. Here, we identify the heterodimerization interface between the α and β subunits of transcription factor GA-binding protein (GAbp) as a negative target of tumor necrosis factor alpha (TNF-α) signaling. TNF-α prevented GAbpα and β complex formation via reactive oxygen species (ROS), leading to the non-energy-dependent transcriptional inactivation of AMP-activated kinase (AMPK) β1, which was identified as a direct hepatic GAbp target. Impairment of AMPKβ1, in turn, elevated downstream cellular cholesterol biosynthesis, and hepatocyte-specific ablation of GAbpα induced systemic hypercholesterolemia and early macro-vascular lesion formation in mice. As GAbpα and AMPKβ1 levels were also found to correlate in obese human patients, the ROS-GAbp-AMPK pathway may represent a key component of a hepato-vascular axis in diabetic long-term complications.

To generate energy efficiently, the cell is uniquely challenged to co-ordinate the abundance of electron transport chain protein subunits expressed from both nuclear and mitochondrial genomes. How an effective stoichiometry of this many constituent subunits is co-ordinated post-transcriptionally remains poorly understood. Here we show that Cerox1, an unusually abundant cytoplasmic long noncoding RNA (lncRNA), modulates the levels of mitochondrial complex I subunit transcripts in a manner that requires binding to microRNA-488-3p. Increased abundance of Cerox1 cooperatively elevates complex I subunit protein abundance and enzymatic activity, decreases reactive oxygen species production, and protects against the complex I inhibitor rotenone. Cerox1 function is conserved across placental mammals: human and mouse orthologues effectively modulate complex I enzymatic activity in mouse and human cells, respectively. Cerox1 is the first lncRNA demonstrated, to our knowledge, to regulate mitochondrial oxidative phosphorylation and, with miR-488-3p, represent novel targets for the modulation of complex I activity.

The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

Mitochondria are complex organelles containing 13 proteins encoded by mitochondrial DNA and over 1,000 proteins encoded on nuclear DNA. Many mitochondrial proteins are associated with the inner or outer mitochondrial membranes, either peripherally or as integral membrane proteins, while others reside in either of the two soluble mitochondrial compartments, the mitochondrial matrix and the intermembrane space. The biogenesis of the five complexes of the oxidative phosphorylation system are exemplars of this complexity. These large multi-subunit complexes are comprised of more than 80 proteins with both membrane integral and peripheral associations and require soluble, membrane integral and peripherally associated assembly factor proteins for their biogenesis. Mutations causing human mitochondrial disease can lead to defective complex assembly due to the loss or altered function of the affected protein and subsequent destabilization of its interactors. Here we couple sodium carbonate extraction with quantitative mass spectrometry (SCE-MS) to track changes in the membrane association of the mitochondrial proteome across multiple human knockout cell lines. In addition to identifying the membrane association status of over 840 human mitochondrial proteins, we show how SCE-MS can be used to understand the impacts of defective complex assembly on protein solubility, giving insights into how specific subunits and sub-complexes become destabilized.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

The classical function of human chorionic gonadotropin (hCG) is its role in supporting pregnancy. hCG is a dimer consisting of two highly glycosylated subunits, alpha (CGA) and beta (CGB). The beta-hCG protein is encoded by CGB3, CGB5, CGB7 and CGB8 genes. CGB3, 5 and 8 code for an identical protein, CGB3/5/8, whereas CGB7 differs in three amino acids from CGB3/5/8. We had observed earlier that CGB7 and CGB3/5/8 display very distinct tissue expression patterns and that the tumor suppressor and transcription factor p53 can activate expression of CGB7 but not of CGB3/5/8 genes. Here, we investigate the glycan structures and possible functional differences of the two CGB variants. To this end, we established a system to produce and isolate recombinant CGA, CGB7 and CGB3/5/8 proteins. We found that N- and O-glycosylation patterns of CGB7 and CGB3/5/8 are quite similar. Functional assays were performed by testing activation of the ERK1/2 pathway and demonstrated that CGB7 and CGB5/5/8 appear to be functionally redundant isoforms, although a slight difference in the kinetics of ERK1/2 pathway activation was observed. This is the first time that biological activity of CGB7 is shown. In summary, the results lead to the hypothesis that CGB7 and CGB3/5/8 do not hold significant functional differences but that timing and cell type of their expression is the key for understanding their divergent evolution.

The Escherichia coli aer regulatory region contains a single promoter that is recognized by RNA polymerase containing the flagellar sigma factor, sigma(28). Expression from this promoter is dependent on direct activation by the cyclic AMP receptor protein, which binds to a target centred 49.5 base pairs upstream from the transcript start. Activator-dependent transcription from the aer promoter was reconstituted in vitro, and a tethered inorganic nuclease was used to find the position of the C-terminal domains of the RNA polymerase alpha subunits in transcriptionally competent open complexes. We report that the ternary activator--RNA polymerase--aer promoter open complex is organized differently from complexes at previously characterized promoters. Among other E. coli promoters recognized by RNA polymerase containing sigma(28), only the trg promoter is activated directly by the cyclic AMP receptor protein. The organization of the different promoter elements and the activator binding site at the trg promoter is the same as at the aer promoter, suggesting a common activation mechanism.

Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.