Carboxy-terminal Domain Research Articles

Expression and copper binding characteristics of Plasmodium falciparum cytochrome c oxidase assembly factor 11, Cox11

BackgroundCopper is an essential metal for living organisms as a catalytic co-factor for important enzymes, like cytochrome c oxidase the final enzyme in the electron transport chain. Plasmodium falciparum parasites in infected red blood cells are killed by excess copper and development in erythrocytes is inhibited by copper chelators. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11.MethodsA 162 amino acid carboxy-terminal domain of the P. falciparum Cox11 ortholog (PfCox11Ct) was recombinantly expressed and the rMBPPfCox11Ct affinity purified. Copper binding was measured in vitro and in Escherichia coli host cells. Site directed mutagenesis was used to identify key copper binding cysteines. Antibodies confirmed the expression of the native protein.ResultsrMBPPfCox11Ct was expressed as a 62 kDa protein fused with the maltose binding protein and affinity purified. rMBPPfCox11Ct bound copper measured by: a bicinchoninic acid release assay; atomic absorption spectroscopy; a bacterial host growth inhibition assay; ascorbate oxidation inhibition and in a thermal shift assay. The cysteine 157 amino acid was shown to be important for in vitro copper binding by PfCox11whilst Cys 60 was not. The native protein was detected by antibodies against rMBPPfCox11Ct.ConclusionsPlasmodium spp. express the PfCox11 protein which shares structural features and copper binding motifs with Cox11 from other species. PfCox11 binds copper and is, therefore, predicted to transfer copper to the CuB site of Plasmodium cytochrome c oxidase. Characterization of Plasmodium spp. proteins involved in copper metabolism will help sceintists understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.

Comprehensive evaluation and efficient classification of BRCA1 RING domain missense substitutions

BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein-truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a mammalian two-hybrid assay. Downstream of the laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data, such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that 15%-20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 45 with concordant computational and functional assay evidence in favor of pathogenicity plus 223 with concordant evidence in favor of benignity; these are particularly likely to be classified as likely pathogenic and likely benign, respectively, once human observational data become available.

Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes.

Gasdermins (GSDMs) are a family of pore-forming effectors that permeabilize the cell membrane during the cell death program pyroptosis1. GSDMs are activated by proteolytic removal of autoinhibitory carboxy-terminal domains, typically by caspase regulators1-9. However, no activator is known for one member of this family, GSDMA. Here we show that the major human pathogen group A Streptococcus (GAS) secretes a protease virulence factor, SpeB, that induces GSDMA-dependent pyroptosis. SpeB cleavage of GSDMA releases an active amino-terminal fragment that can insert into membranes to form lytic pores. GSDMA is primarily expressed in the skin10, and keratinocytes infected with SpeB-expressing GAS die of GSDMA-dependent pyroptosis. Mice have three homologues of human GSDMA, and triple-knockout mice are more susceptible to invasive infection by a pandemic hypervirulent M1T1 clone of GAS. These results indicate that GSDMA is critical in the immune defence against invasive skin infections by GAS. Furthermore, they show that GSDMs can act independently of host regulators as direct sensors of exogenous proteases. As SpeB is essential for tissue invasion and survival within skin cells, these results suggest that GSDMA can act akin to a guard protein that directly detects concerning virulence activities of microorganisms that present a severe infectious threat.

The C-terminal domain of Arabidopsis ROS1 DNA demethylase interacts with histone H3 and is required for DNA binding and catalytic activity

Active DNA demethylation plays an important role in controlling methylation patterns in eukaryotes. In plants, the DEMETER-LIKE (DML) family of 5-methylcytosine DNA glycosylases initiates DNA demethylation through a base excision repair pathway. However, it is poorly understood how these DNA demethylases are recruited to their target loci and the role that histone marks play in this process. Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) is a representative enzyme of the DML family, whose members are uniquely characterized by a basic amino-terminal domain mediating nonspecific binding to DNA, a discontinuous catalytic domain, and a conserved carboxy-terminal domain of unknown function. Here, we show that ROS1 interacts with the N-terminal tail of H3 through its C-terminal domain. Importantly, phosphorylation at H3 Ser28, but not Ser10, abrogates ROS1 interaction with H3. Conserved residues at the C-terminal domain are not only required for H3 interaction, but also for efficient DNA binding and catalytic activity. Our findings suggest that the C-terminal domain of ROS1 may function as a histone reader module involved in recruitment of the DNA demethylase activity to specific genomic regions.

FC 132: Deficiency of Proximal Tubular Cyclin-Dependent Kinase 12 Exacerbates Kidney Injury Through DNA-Damage Response

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is a frequent, serious condition characterized by acute tubular necrosis involving the destruction of tubular epithelial cells. Recent studies have shown that DNA damage contributes to renal tubular cell death during kidney injury. Cyclin-dependent kinase 12 (CDK12) is a member of the cyclin-dependent kinase family that phosphorylates the carboxy-terminal domain of RNA polymerase II and selectively regulates transcription of DNA-damage response (DDR) genes. CDK12 inhibitor was reported to play an important role in a variety of cancers. So far, the functions of CDK12 deficiency in the pathogenesis of AKI have not been studied. METHOD Here, we established a cisplatin-induced AKI mouse model with CDK12 knockdown mice and created stable in vitro models with CDK12 knockdown tubular epithelial cells. Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (20 mg/kg). After 2 days following cisplatin treatment, blood and kidney tissues were harvested. Renal function and histology were evaluated. DDR, tubular apoptosis, cell proliferation were evaluated by immunofluorescence assays, real-time PCR, flow cytometer and western blot analysis. RESULTS Here we found that CDK12 was downregulated in the renal tubular epithelial cells of AKI patients and murine AKI models and associated with kidney functions. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in DDR and tubular apoptosis signaling pathways and decreased transcripts cell proliferation pathways, in knockout versus control mice. Moreover, more severe AKI occurred in tubular cell-specific knock-down of CDK12 mice (CDK12RTEC+/–) than age- and sex-matched wild type mice, as evidenced by increased levels of serum creatinine (Scr), blood urea nitrogen (BUN) and tubular injury score. Mechanistically, CDK12 deficiency accelerated cisplatin-induced DDR, mitochondrial apoptosis and abrogates cell proliferation of renal tubular cells in these mice. Similar results were observed after the human renal tubular epithelial cell line HK-2 cells exposure to CDK12 siRNA stimulation. In contrast, lentivirus-mediated CDK12 overexpression protected against DDR and mitochondrial apoptosis while attenuating renal dysfunction, morphological damage and renal tubular cell death in cisplatin-induced AKI models. Moreover, representative Integrative Genomics Viewer (IGV) profiles demonstrated a minimal impact of gene length-dependent elongation defects, a substantial proportion of which participate in the DDR and cell proliferation. CONCLUSION Thus, proximal tubule-specific deficiency of CDK12 exacerbates acute kidney injury and tubular apoptosis, likely due to affecting the expression of genes involved in the DDR and mRNA processing.

Structure of cysteate acyltransferase, the condensation enzyme that initiates sulfonolipid biosynthesis by inhabitants of the gut microbiome

Sulfonolipids (SL) are unusual lipids found in the outer membranes of Gram‐negative bacteria in the phylum Bacteroidetes. SL and its deacylated derivative, capnine, are sulfur analogs of ceramide‐1‐phosphate and sphingosine‐1‐phosphate, respectively. The structural similarity to key mammalian signaling molecules led us to investigate the biochemistry and distribution of SL production in inhabitants of the gut microbiome. Alistipes finegoldii, an anaerobic gut commensal contains two genes related to the bacterial serine palmitoyl transferase (Spt) identified in Sphingobacteriia. The two proteins were purified and the Afl_1224 gene product catalyzed the condensation of [14C]acyl‐acyl carrier protein and cysteate (3‐sulfo‐alanine). The product of cysteate acyltransferase (AfCat) was identified as 3‐ketocapnine by mass spectrometry. AfCat has a bound pyridoxal phosphate cofactor (PLP) that undergoes a spectral blue shift when exposed to cysteate consistent with the transition of a lysine‐aldimine complex to a substrate‐aldimine complex. We solved the crystal structure of dimeric AfCat using SAD phasing. AfCat has the prototypical fold enveloping the PLP cofactor bound to Lys‐251 found in Spt and has an electrostatic surface patch for ACP binding. AfCat is distinguished from Spt by its carboxy terminal domain that extends to interact with the amino terminus, an enlarged substrate binding site to accommodate and stabilize the negatively charged sulfono head group of cysteate, and the HAS motif in Spt is HAC in AfCat. These unique structural elements were used to identify SL producing organisms in metagenomic sequencing data from the gut microbiome that are responsible for SL and capnine in mammalian stool samples. Supported by GM034496.

Atypical Antibody Dynamics During Human Coronavirus HKU1 Infections

Human coronavirus HKU1 (HCoV-HKU1) is one of the four endemic coronaviruses. It has been suggested that there is a difference in incidence, with PCR-confirmed HCoV-NL63 and HCoV-OC43 infections occurring more commonly, whereas HCoV-HKU1 is the least seen. Lower incidence of HCoV-HKU1 infection has also been observed in serological studies. The current study aimed to investigate antibody dynamics during PCR-confirmed HCoV-HKU1 infections using serum collected during infection and 1 month later. We expressed a new HCoV-HKU1 antigen consisting of both the linker and carboxy-terminal domain of the viral nucleocapsid protein and implemented it in ELISA. We also applied a spike-based Luminex assay on serum samples from PCR-confirmed infections by the four endemic HCoVs. At least half of HCoV-HKU1-infected subjects consistently showed no antibody rise via either assay, and some subjects even exhibited substantial antibody decline. Investigation of self-reported symptoms revealed that HCoV-HKU1-infected subjects rated their illness milder than subjects infected by other HCoVs. In conclusion, HCoV-HKU1 infections reported in this study displayed atypical antibody dynamics and milder symptoms when compared to the other endemic HCoVs.

TDP-43 Oligomerization and Phase Separation Properties Are Necessary for Autoregulation.

Loss of TDP-43 protein homeostasis and dysfunction, in particular TDP-43 aggregation, are tied to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is an RNA binding protein tightly controlling its own expression levels through a negative feedback loop, involving TDP-43 recruitment to the 3′ untranslated region of its own transcript. Aberrant TDP-43 expression caused by autoregulation defects are linked to TDP-43 pathology. Therefore, interactions between TDP-43 and its own transcript are crucial to prevent TDP-43 aggregation and loss of function. However, the mechanisms that mediate this interaction remain ill-defined. We find that a central RNA sequence in the 3′ UTR, which mediates TDP-43 autoregulation, increases the liquid properties of TDP-43 phase separation. Furthermore, binding to this RNA sequence induces TDP-43 condensation in human cell lysates, suggesting that this interaction promotes TDP-43 self-assembly into dynamic ribonucleoprotein granules. In agreement with these findings, our experiments show that TDP-43 oligomerization and phase separation, mediated by the amino and carboxy-terminal domains, respectively, are essential for TDP-43 autoregulation. According to our additional observations, CLIP34-associated phase separation and autoregulation may be efficiently controlled by phosphorylation of the N-terminal domain. Importantly, we find that specific ALS-associated TDP-43 mutations, mainly M337V, and a shortened TDP-43 isoform recently tied to motor neuron toxicity in ALS, disrupt the liquid properties of TDP-43-RNA condensates as well as autoregulatory function. In addition, we find that M337V decreases the cellular clearance of TDP-43 and other RNA binding proteins associated with ALS/FTD. These observations suggest that loss of liquid properties in M337V condensates strongly affects protein homeostasis. Together, this work provides evidence for the central role of TDP-43 oligomerization and liquid-liquid phase separation linked to RNA binding in autoregulation. These mechanisms may be impaired by TDP-43 disease variants and controlled by specific cellular signaling.

An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

BackgroundUnphosphorylated signal transducer and activator of transcription 1 (U-STAT1) has been reported to elicit a distinct gene expression profile as compared to tyrosine-phosphorylated STAT1 (P-STAT1) homodimers. However, the impact of U-STAT1 on the IFNγ-induced immune response mediated by P-STAT1 is unknown. By generating a double mutant of STAT1 with mutation R602L in the Src-homology 2 (SH2) domain and Y701F in the carboxy-terminal transactivation domain mimicking U-STAT1, we investigated the effects of U-STAT1 on P-STAT1-mediated signal transduction.ResultsIn this study, we discovered a novel activity of U-STAT1 that alters the nucleo-cytoplasmic distribution of cytokine-stimulated P-STAT1. While the dimerization-deficient mutant R602L/Y701F was not able to display cytokine-induced nuclear accumulation, it inhibited the nuclear accumulation of co-expressed IFNγ-stimulated wild-type P-STAT1. Disruption of the anti-parallel dimer interface in the R602L/Y701F mutant via additional R274W and T385A mutations did not rescue the impaired nuclear accumulation of co-expressed P-STAT1. The mutant U-STAT1 affected neither the binding of co-expressed P-STAT1 to gamma-activated sites in vitro, nor the transcription of reporter constructs and the activation of STAT1 target genes. However, the nuclear accumulation of P-STAT1 was diminished in the presence of mutant U-STAT1, which was not restored by mutations reducing the DNA affinity of mutant U-STAT1. Whereas single mutations in the amino-terminus of dimerization-deficient U-STAT1 similarly inhibited the nuclear accumulation of co-expressed P-STAT1, a complete deletion of the amino-terminus restored cytokine-stimulated nuclear accumulation of P-STAT1. Likewise, the disruption of a dimer-specific nuclear localization signal also rescued the U-STAT1-mediated inhibition of P-STAT1 nuclear accumulation.ConclusionOur data demonstrate a novel role of U-STAT1 in affecting nuclear accumulation of P-STAT1, such that a high intracellular concentration of U-STAT1 inhibits the detection of nuclear P-STAT1 in immunofluorescence assays. These observations hint at a possible physiological function of U-STAT1 in buffering the nuclear import of P-STAT1, while preserving IFNγ-induced gene expression. Based on these results, we propose a model of a hypothetical import structure, the assembly of which is impaired under high concentrations of U-STAT1. This mechanism maintains high levels of cytoplasmic STAT1, while simultaneously retaining signal transduction by IFNγ.4WV28nXUESFNnmyyzU2Dx5Video

Unveiling RCOR1 as a rheostat at transcriptionally permissive chromatin

RCOR1 is a known transcription repressor that recruits and positions LSD1 and HDAC1/2 on chromatin to erase histone methylation and acetylation. However, there is currently an incomplete understanding of RCOR1’s range of localization and function. Here, we probe RCOR1’s distribution on a genome-wide scale and unexpectedly find that RCOR1 is predominantly associated with transcriptionally active genes. Biochemical analysis reveals that RCOR1 associates with RNA Polymerase II (POL-II) during transcription and deacetylates its carboxy-terminal domain (CTD) at lysine 7. We provide evidence that this non-canonical RCOR1 activity is linked to dampening of POL-II productive elongation at actively transcribing genes. Thus, RCOR1 represses transcription in two ways—first, via a canonical mechanism by erasing transcriptionally permissive histone modifications through associating with HDACs and, second, via a non-canonical mechanism that deacetylates RNA POL-II’s CTD to inhibit productive elongation. We conclude that RCOR1 is a transcription rheostat.

Antitumor Efficacy of EGFR-Targeted Recombinant Immunotoxin in Human Head and Neck Squamous Cell Carcinoma.

Over 90% of head and neck squamous cell carcinoma (HNSCC) overexpresses the epidermal growth factor receptor (EGFR). However, the EGFR-targeted monotherapy response rate only achieves 10-30% in HNSCC. Recombinant immunotoxin (RIT) often consists of an antibody targeting a tumor antigen and a toxin (e.g., diphtheria toxin [DT]) that kills cancer cells. We produced a humanized RIT, designated as hDT806, targeting overexpressed EGFR and investigated its effects in HNSCC. Distinct from the EGFR-targeted tyrosine kinase inhibitor erlotinib or antibody cetuximab, hDT806 effectively suppressed cell proliferation in the four HNSCC lines tested (JHU-011, -013, -022, and -029). In JHU-029 mouse xenograft models, hDT806 substantially reduced tumor growth. hDT806 decreased EGFR protein levels and disrupted the EGFR signaling downstream effectors, including MAPK/ERK1/2 and AKT, while increased proapoptotic proteins, such as p53, caspase-9, caspase-3, and the cleaved PAPR. The hDT806-induced apoptosis of HNSCC cells was corroborated by flow cytometric analysis. Furthermore, hDT806 resulted in a drastic inhibition in RNA polymerase II carboxy-terminal domain phosphorylation critical for transcription and a significant increase in the γH2A.X level, a DNA damage marker. Thus, the direct disruption of EGFR signaling, transcription inhibition, DNA damage, as well as apoptosis induced by hDT806 may contribute to its antitumor efficacy in HNSCC.

Structure of a Janus kinase cytokine receptor complex reveals the basis for dimeric activation.

Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus kinases (JAKs). We report the 3.6-angstrom-resolution cryo-electron microscopy structure of full-length JAK1 complexed with a cytokine receptor intracellular domain Box1 and Box2 regions captured as an activated homodimer bearing the valine→phenylalanine (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit, the dimerization of which is mediated by close-packing of the pseudokinase (PK) domains from the monomeric subunits. The oncogenic VF mutation lies within the core of the JAK1 PK interdimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The carboxy-terminal tyrosine kinase domains are poised for transactivation and to phosphorylate the receptor STAT (signal transducer and activator of transcription)-recruiting motifs projecting from the overhanging FERM (four-point-one, ezrin, radixin, moesin)-SH2 (Src homology 2)-domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals opportunities for selective therapeutic targeting of oncogenic JAK signaling.

Dystrophic Cardiomyopathy and the Need for Cardiovascular Care

Duchenne muscular dystrophy (DMD) affects approximately 1 in 5,000 boys worldwide.1,2 This X-linked recessive disease is a result of spontaneous or inherited mutations in the dystrophin gene.3 The dystrophin gene is the longest gene in the human genome as it spans 2.5 megabases, includes 79 exons, and encodes a 427-kDa cytolinker protein.1,3 The dystrophin protein connects the interior of the cell (skeletal myocyte, cardiomyocyte, or neuron) by binding to actin via its amino terminal domain to the sarcolemma as it binds to β-dystroglycan via its carboxyterminal domain.

Transcription by RNA polymerase II and the CTD-chromatin crosstalk

The epigenetic phenomenon is known to derive the phenotypic variation of an organism through an interconnected cellular network of histone modifications, DNA methylation and RNA regulatory network. Transcription for protein coding genes is a highly regulated process and carried out by a large multi-complex RNA Polymerase II. The carboxy terminal domain (CTD) of the largest subunit of RNA Polymerase II consists of a conserved and highly repetitive heptad sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The epigenetically modified CTD is thought to selectively bind different protein complexes that participate in mRNA biogenesis and export. The CTD and chromatin appears to have a spatial relationship during the transcription cycle, where the epigenetic modifications of CTD not only influence the state of histone modification but also mediates CTD-chromatin crosstalk. In this mini review, we have surveyed and discussed current developments of RNA Polymerase II CTD and its new emerging crosstalk with chromatin, during the stage specific progression of RNA Polymerase II in transcription cycle. This review is mainly focussed on the insights in budding yeast.

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and β1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

Truncations of Nav1.5 carboxy-terminal domain differentially perturb channel function

The voltage-gated sodium channel (NaV1.5) is critical for cardiac function. Rapid activation of NaV1.5 supports the initiation of the action potential, while ensuing inactivation is essential for proper repolarization. Human channelopathic mutations in NaV1.5 have been linked to various cardiac maladies, including cardiac arrhythmia, atrial fibrillation, and dilated cardiomyopathies. The carboxy-terminal domain (CTD) of NaV1.5 is critical for fine-tuning channel inactivation and is well-recognized as a hotspot for channelopathic mutations.

The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.

Carboxy-terminal domain (CTD) small phosphatase like 2 (CTDSPL2), also known as SCP4 or HSPC129, is a new member of the small CTD phosphatase (SCP) family and its role in cancers remains unclear. Here, we used a Phos-tag technique to screen a series of phosphatases and identified CTDSPL2 as a mitotic regulator. We demonstrated that CTDSPL2 was phosphorylated at T86, S104, and S134 by cyclin-dependent kinase 1 (CDK1) in mitosis. Depletion of CTDSPL2 led to mitotic defects and prolonged mitosis. Resultantly, CTDSPL2 deletion restrained proliferation, migration, and invasion in pancreatic cancer cells. We further confirmed the dominant negative effects of a phosphorylation-deficient mutant form of CTDSPL2, implying the biological significance of CTDSPL2 mitotic phosphorylation. Moreover, RT2 cell cycle array analysis revealed p21 and p27 as downstream regulators of CTDSPL2, and inhibition of p21 and/or p27 partially rescued the phenotype in CTDSPL2-deficient cell lines. Importantly, both CTDSPL2 depletion and phosphorylation-deficient mutant CTDSPL2 hindered tumor growth in xenograft models. Together, our findings for the first time highlight the novel role of CTDSPL2 in regulating cell mitosis, proliferation and motility in pancreatic cancer and point out the implications of CTDSPL2 in regulating two critical cell cycle participants (p21 and p27), providing an alternative molecular target for pancreatic cancer treatment.

Visualization, Quantification, and Modeling of Endogenous RNA Polymerase II Phosphorylation at a Single-copy Gene in Living Cells.

In eukaryotic cells, RNA Polymerase II (RNAP2) is the enzyme in charge of transcribing mRNA from DNA. RNAP2 possesses an extended carboxy-terminal domain (CTD) that gets dynamically phosphorylated as RNAP2 progresses through the transcription cycle, therefore regulating each step of transcription from recruitment to termination. Although RNAP2 residue-specific phosphorylation has been characterized in fixed cells by immunoprecipitation-based assays, or in live cells by using tandem gene arrays, these assays can mask heterogeneity and limit temporal and spatial resolution. Our protocol employs multi-colored complementary fluorescent antibody-based (Fab) probes to specifically detect the CTD of the RNAP2 (CTD-RNAP2), and its phosphorylated form at the serine 5 residue (Ser5ph-RNAP2) at a single-copy HIV-1 reporter gene. Together with high-resolution fluorescence microscopy, single-molecule tracking analysis, and rigorous computational modeling, our system allows us to visualize, quantify, and predict endogenous RNAP2 phosphorylation dynamics and mRNA synthesis at a single-copy gene, in living cells, and throughout the transcription cycle. Graphical abstract: Schematic of the steps for visualizing, quantifying, and predicting RNAP2 phosphorylation at a single-copy gene.

Apolipoprotein A-I carboxy-terminal domain residues 187–243 are required for adiponectin-induced cholesterol efflux

Adiponectin exerts its atheroprotection by stimulating adenosine triphosphate binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). However, involvement of the apoA-I residues in this process have not been studied. In Tamm-Horsfall 1 (THP-1) macrophages and baby hamster kidney (BHK) cells we assessed adiponectin's potential to restore cholesterol efflux in the presence of apoA-I and ABCA1 mutants, respectively. Adiponectin was unable to restore efflux from THP-1 macrophages in the presence of apoA-I carboxy-terminal domain (CTD) successive mutants from residues 187–243 versus apoA-I mutants alone. Furthermore, adiponectin did not significantly influence cholesterol efflux to apoA-I from BHK-ABCA1 mutant cells. Adiponectin appears to require functional apoA-I CTD residues 187–243 and wild-type ABCA1 to mediate efficient cholesterol efflux from THP-1 macrophages and BHK cells, respectively. Therefore, adiponectin cannot rescue defective cholesterol efflux in apoA-I- or ABCA1-mutant conditions, but rather increases cholesterol efflux in wild-type apoA-I conditions compared to apoA-I exposure alone.

Structural architecture of the human NALCN channelosome.

Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1-8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9-12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.