Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IκBα through a phosphoenzyme intermediate.

Rapid and high-fidelity phosphorylation of two serines (S32 and S36) of IκBα by a prototype Ser/Thr kinase IKK2 is critical for fruitful canonical NF-κB activation. Here, we report that IKK2 is a dual specificity Ser/Thr kinase that autophosphorylates itself at tyrosine residues in addition to its activation loop serines. Mutation of one such tyrosine, Y169, located in proximity to the active site, to phenylalanine, renders IKK2 inactive for phosphorylation of S32 of IκBα. Surprisingly, auto-phosphorylated IKK2 relayed phosphate group(s) to IκBα without ATP when ADP is present. We also observed that mutation of K44, an ATP-binding lysine conserved in all protein kinases, to methionine renders IKK2 inactive towards specific phosphorylation of S32 or S36 of IκBα, but not non-specific substrates. These observations highlight an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop and the invariant ATP-binding K44 residue define its signal-responsive substrate specificity ensuring the fidelity of NF-κB activation.

A glycolytic metabolite restores DNA repair activity of polynucleotide kinase 3'-phosphatase in polyglutamine (PolyQ) diseases.

We previously reported that the loss of activity of an essential DNA repair enzyme, polynucleotide kinase 3'-phosphatase (PNKP), resulted in accumulation of double strand breaks (DSB) in patient's brain genome in Huntington's disease (HD) and Spinocerebellar ataxia type 3 (SCA3). Here we document that PNKP interacts with the nuclear isoform of phosphofructokinase fructose-2,6-bisphosphatase 3 (PFKFB3), which converts fructose-6-phosphate (F6P) into fructose-2,6-bisphosphate (F2,6BP), a potent allosteric modulator of glycolysis. Depletion of PFKFB3 markedly abrogates PNKP activity, thereby affecting PNKP mediated transcription-coupled non-homologous end joining (TC-NHEJ). Both PFKFB3 and F2,6BP levels are significantly lower in the nuclear extracts of HD and SCA3 patients' brains. Exogenous F2,6BP restored PNKP activity in the brain nuclear extracts of those samples. Moreover, delivery of F2,6BP into HD mouse striata-derived neuronal cells restored PNKP activity, transcribed genome integrity and cellular viability. We thus postulate that F2,6BP serves in vivo as a cofactor for proper functionality of PNKP and thereby of brain health. Our results thus provide a compelling rationale for exploring therapeutic use of F2,6BP and related compounds for treating polyQ diseases.

Structural Requirements for Reverse Transcription by a Diversity-generating Retroelement.

Diversity-generating retroelements (DGRs), which are pervasive among microbes, create massive protein sequence variation through reverse transcription of a protein-coding RNA template coupled to frequent misincorporation at template adenines. For cDNA synthesis, the template must be surrounded by up- and downstream sequences. Cryo-EM revealed that this longer RNA formed an integral ribonucleoprotein (RNP) with the DGR reverse transcriptase bRT and associated protein Avd. The downstream, noncoding (nc) RNA formed stem-loops enveloping bRT and laying over barrel-shaped Avd, and duplexes with the upstream and template RNA. These RNA structural elements were required for reverse transcription, and several were conserved in DGRs from distant taxa. Multiple RNP conformations were visualized, and no large structural rearrangements occurred when adenine replaced guanine as the template base, suggesting energetics govern misincorporation at adenines. Our results explain how the downstream ncRNA primes cDNA synthesis, promotes processivity, terminates polymerization, and stringently limits mutagenesis to DGR variable proteins.

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2.

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKß subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKß, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the "NEMO-binding domain" at the C terminus of IKK2/IKKß. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKß through this secondary interaction in vitro and for full activation of IKK2/IKKß in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKß within its scaffold-dimerization domain proximal to the kinase domain-Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture-based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKß for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.

Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code.

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of 3D structural scaffold of AdML-a model pre-mRNA substrate-guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a 3D structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable 3D structural scaffolds to the splicing code across the mammalian transcriptome.

Structurally plastic NEMO and oligomerization prone IKK2 subunits define the behavior of human IKK2:NEMO complexes in solution.

The human IκB Kinase (IKK) is a multisubunit protein complex of two kinases and one scaffolding subunit that controls induction of transcription factor NF-κB activity. IKK behaves as an entity of aberrantly high apparent molecular weight in solution. Recent X-ray crystallographic and cryo-electron microscopy structures of individual catalytic subunits (IKK1/IKKα and IKK2/IKKß) reveal that they are both stably folded dimeric proteins that engage in extensive homo-oligomerization through unique surfaces that are required for activation of their respective catalytic activities. The NEMO/IKKγ subunit is a predominantly coiled coil protein that is required for activation of IKK through the canonical NF-κB signaling pathway. Here we report size-exclusion chromatography, multi-angle light scattering, analytical centrifugation, and thermal denaturation analyses of full-length human recombinant NEMO as well as deletion and disease-linked variants. We observe that NEMO is predominantly a dimer in solution, although by virtue of its modular coiled coil regions NEMO exhibits complicated solution dynamics involving portions that are mutually antagonistic toward homodimerization. This behavior causes NEMO to behave as a significantly larger sized particle in solution. Analyses of NEMO in complex with IKK2 indicate that NEMO preserves this structurally dynamic character within the multisubuit complex and provides the complex-bound IKK2 further propensity toward homo-oligomerization. These observations provide critical information on the structural plasticity of NEMO subunit dimers which helps clarify its role in diseases and in IKK regulation through oligomerization-dependent phosphorylation of catalytic IKK2 subunit dimers.

CD1d Selectively Down Regulates the Expression of the Oxidized Phospholipid-Specific E06 IgM Natural Antibody in Ldlr-/- Mice.

Natural antibodies (NAbs) are important regulators of tissue homeostasis and inflammation and are thought to have diverse protective roles in a variety of pathological states. E06 is a T15 idiotype IgM NAb exclusively produced by B-1 cells, which recognizes the phosphocholine (PC) head group in oxidized phospholipids on the surface of apoptotic cells and in oxidized LDL (OxLDL), and the PC present on the cell wall of Streptococcus pneumoniae. Here we report that titers of the E06 NAb are selectively increased several-fold in Cd1d-deficient mice, whereas total IgM and IgM antibodies recognizing other oxidation specific epitopes such as in malondialdehyde-modified LDL (MDA-LDL) and OxLDL were not increased. The high titers of E06 in Cd1d-deficient mice are not due to a global increase in IgM-secreting B-1 cells, but they are specifically due to an expansion of E06-secreting splenic B-1 cells. Thus, CD1d-mediated regulation appeared to be suppressive in nature and specific for E06 IgM-secreting cells. The CD1d-mediated regulation of the E06 NAb generation is a novel mechanism that regulates the production of this specific oxidation epitope recognizing NAb.

Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing.

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

Nuclear magnetic resonance spectroscopy of human body fluids and in vivo magnetic resonance spectroscopy: Potential role in the diagnosis and management of prostate cancer.

Prostate cancer is the most common solid organ cancer in men, and the second most common cause of male cancer-related mortality. It has few effective therapies, and is difficult to diagnose accurately. Prostate-specific antigen (PSA), which is currently the most effective diagnostic tool available, cannot reliably discriminate between different pathologies, and in fact only around 30% of patients found to have elevated levels of PSA are subsequently confirmed to actually have prostate cancer. As such, there is a desperate need for more reliable diagnostic tools that will allow the early detection of prostate cancer so that the appropriate interventions can be applied. Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance spectroscopy (MRS) are 2 high throughput, noninvasive analytical procedures that have the potential to enable differentiation of prostate cancer from other pathologies using metabolomics, by focusing specifically on certain metabolites which are associated with the development of prostate cancer cells and its progression. The value that this type of approach has for the early detection, diagnosis, prognosis, and personalized treatment of prostate cancer is becoming increasingly apparent. Recent years have seen many promising developments in the fields of NMR spectroscopy and MRS, with improvements having been made to hardware as well as to techniques associated with the acquisition, processing, and analysis of related data. This review focuses firstly on proton NMR spectroscopy of blood serum, urine, and expressed prostatic secretions in vitro, and then on 1- and 2-dimensional proton MRS of the prostate in vivo. Major advances in these fields and methodological principles of data collection, acquisition, processing, and analysis are described along with some discussion of related challenges, before prospects that proton MRS has for future improvements to the clinical management of prostate cancer are considered.

An Overview of Coronavirus COVID-19 with their Pathogenesis and Risk Assessment of the Disease Utilizing Positive Predictive Value of the Clinical and Laboratory Data.

COVID-19 has created a devastating pandemic, infecting more than 200 countries in its wake, only sparing Antarctica. The virus dissociates ferrous ion from the porphyrin ring of heme of haemoglobin-thus hampering the oxygen and carbon dioxide exchange in the lung and tissue. The toxic effect of ferrous (Fe2+) ions and carbon dioxide causes lung damage giving rise to severe respiratory distress and an often observed clotting disorder. Serum ferritin level is increased along with the rise of serum LDH, d-dimer, serum IL-6 and cardiac troponin. Associated leukocytosis, occasional lymphocytopenia and radiological changes of the lung are the pathological hallmarks of the disease. All these parameters including other clinical data such as age, fever, gender and associated co-morbidities may be used as a Risk Assessment tool for COVID-19 before the report of real-time polymerase chain reaction (RT PCR) is available. A timely intervention can contribute to rescuing millions from an untimely death.

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α.

A class of extracellular stimuli requires activation of IKK1/α to induce generation of an NF-κB subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-κB subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/α primarily remains associated with IKK2/ß and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/α within the larger or the smaller complex pool. Constitutive activity of IKK1/α has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/α, and enable its use as a drug target, we expressed recombinant IKK1/α in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/α in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

Structures of the Catalytic Domain of Bacterial Primase DnaG in Complexes with DNA Provide Insight into Key Priming Events.

Bacterial primase DnaG is an essential nucleic acid polymerase that generates primers for replication of chromosomal DNA. The mechanism of DnaG remains unclear due to the paucity of structural information on DnaG in complexes with other replisome components. Here we report the first crystal structures of noncovalent DnaG-DNA complexes, obtained with the RNA polymerase domain of Mycobacterium tuberculosis DnaG and various DNA ligands. One structure, obtained with ds DNA, reveals interactions with DnaG as it slides on ds DNA and suggests how DnaG binds template for primer synthesis. In another structure, DNA in the active site of DnaG mimics the primer, providing insight into mechanisms for the nucleotide transfer and DNA translocation. In conjunction with the recent cryo-EM structure of the bacteriophage T7 replisome, this study yields a model for primer elongation and hand-off to DNA polymerase.

Acetylation by Eis and Deacetylation by Rv1151c of Mycobacterium tuberculosis HupB: Biochemical and Structural Insight.

Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from Mycobacterium tuberculosis (Mtb) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD+-dependent deacetylase Rv1151c from Mtb deacetylated HupB in vitro and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes.

DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated.

The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association-dissociation processes.

Erratum: Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

This corrects the article DOI: 10.1038/nmicrobiol.2016.155.

Tapioca Cellulose Based Copper Nanoparticles for Chemoselective N-Alkylation.

Biomaterials as a support for catalysts are of prime importance. Tapioca root which is an abundant biopolymer source was used to synthesize cellulose supported bio-heterogeneous poly(hydroxamic acid) copper nanoparticles (CuN@PHA) and was characterized by Fourier transform infrared spectroscopy (FTIR), ultraviolet­visible spectroscopy (UV-Vis), field emission scanning electron microscopy (FESEM), X-ray photoelectron spectroscopy (XPS), inductively coupled plasma atomic emission spectroscopy (ICP-AES), transmission electron microscopy (TEM) analyses. The tapioca cellulose supported CuN@PHA (50 mol ppm) effectively catalyzed N-alkylation reaction of aliphatic amines with α,ß-unsaturated compounds to give the corresponding alkylated products. High yields up to 95% were achieved for the converted products. The reusability of the cellulose supported nanoparticles was found to be excellent with no significant reduction of its catalytic activity over several cycles. The catalyst showed high catalytic activity having turnover number (TON) 18000 and turnover frequency (TOF) 2250 h⁻¹.

Structural Basis for the Activation of IKK1/α.

Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/ß (IKK2/ß), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is a trimer of IKK1 dimers. While IKK1 hexamers are not detectable in cells, the surface that supports hexamer formation is critical for IKK1-dependent cellular processing of p100 to p52, the hallmark of non-canonical NF-κB signaling. Comparison of this surface to that in IKK2 indicates significant divergence, and it suggests a fundamental role for this surface in signaling by these kinases through distinct pathways.

To Swap or Not To Swap.

Domain swapping is a form of protein oligomerization in which identical structural units are exchanged among protomers within an oligomer. In this issue of Structure, Assar et al. (2016) report domain-swapped dimers of hCREBPII, pinpointing a single hydrogen bond and protein concentration as two critical regulators of the monomer/dimer balance.

Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ∼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design.

Synthesis of new liquid crystals embedded gold nanoparticles for photoswitching properties.

A new series of liquid crystals decorated gold nanoparticles is synthesized whose molecular architecture has azobenzenes moieties as the peripheral units connected to gold nanoparticles (Au NPs) via alkyl groups. The morphology and mesomorphic properties were investigated by field emission scanning electron microscope, high-resolution transmission electron microscopy, differential scanning calorimetry and polarizing optical microscopy. The thiolated ligand molecules (3a-c) showed enantiotropic smectic A phase, whereas gold nanoparticles (5a-c) exhibit nematic and smectic A phase with monotropic nature. HR-TEM measurement showed that the functionalized Au NPs are of the average size of 2nm and they are well dispersed without any aggregation. The trans-form of azo compounds showed a strong band in the UV region at ∼378nm for the π-π(∗) transition, and a weak band in the visible region at ∼472nm due to the n-π(∗) transition. These molecules exhibit attractive photoisomerization behaviour in which trans-cis transition takes about 15s whereas the cis-trans transition requires about 45min for compound 5c. The extent of reversible isomerization did not decay after 10 cycles, which proved that the photo-responsive properties of 5c were stable and repeatable. Therefore, these materials may be suitably exploited in the field of molecular switches and the optical storage devices.