Structures of human TR4LBD-JAZF1 and TR4DBD-DNA complexes reveal the molecular basis of transcriptional regulation.

Testicular nuclear receptor 4 (TR4) modulates the transcriptional activation of genes and plays important roles in many diseases. The regulation of TR4 on target genes involves direct interactions with DNA molecules via the DNA-binding domain (DBD) and recruitment of coregulators by the ligand-binding domain (LBD). However, their regulatory mechanisms are unclear. Here, we report high-resolution crystal structures of TR4DBD, TR4DBD-DNA complexes and the TR4LBD-JAZF1 complex. For DNA recognition, multiple factors come into play, and a specific mutual selectivity between TR4 and target genes is found. The coactivators SRC-1 and CREBBP can bind at the interface of TR4 originally occupied by the TR4 activation function region 2 (AF-2); however, JAZF1 suppresses the binding through a novel mechanism. JAZF1 binds to an unidentified surface of TR4 and stabilizes an α13 helix never reported in the nuclear receptor family. Moreover, the cancer-associated mutations affect the interactions and the transcriptional activation of TR4 in vitro and in vivo, respectively. Overall, our results highlight the crucial role of DNA recognition and a novel mechanism of how JAZF1 reinforces the autorepressed conformation and influences the transcriptional activation of TR4, laying out important structural bases for drug design for a variety of diseases, including diabetes and cancers.

Structural Basis of Nucleic Acid Recognition and 6mA Demethylation by Caenorhabditis elegans NMAD-1A.

N6-methyladenine (6mA) of DNA is an emerging epigenetic mark in the genomes of Chlamydomonas, Caenorhabditis elegans, and mammals recently. Levels of 6mA undergo drastic fluctuation and thus affect fertility during meiosis and early embryogenesis. Here, we showed three complex structures of 6mA demethylase C. elegans NMAD-1A, a canonical isoform of NMAD-1 (F09F7.7). Biochemical results revealed that NMAD-1A prefers 6mA Bubble or Bulge DNAs. Structural studies of NMAD-1A revealed an unexpected "stretch-out" conformation of its Flip2 region, a conserved element that is usually bent over the catalytic center to facilitate substrate base flipping in other DNA demethylases. Moreover, the wide channel between the Flip1 and Flip2 of the NMAD-1A explained the observed preference of NMAD-1A for unpairing substrates, of which the flipped 6mA was primed for catalysis. Structural analysis and mutagenesis studies confirmed that key elements such as carboxy-terminal domain (CTD) and hypothetical zinc finger domain (ZFD) critically contributed to structural integrity, catalytic activity, and nucleosome binding. Collectively, our biochemical and structural studies suggest that NMAD-1A prefers to regulate 6mA in the unpairing regions and is thus possibly associated with dynamic chromosome regulation and meiosis regulation.

Structural insights into the interactions and epigenetic functions of human nucleic acid repair protein ALKBH6.

Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy.

Structural Basis of Human Parainfluenza Virus 3 Unassembled Nucleoprotein in Complex with Its Viral Chaperone.

Human parainfluenza virus 3 (HPIV3) belongs to the Paramyxoviridae, causing annual worldwide epidemics of respiratory diseases, especially in newborns and infants. The core components consist of just three viral proteins: nucleoprotein (N), phosphoprotein (P), and RNA polymerase (L), playing essential roles in replication and transcription of HPIV3 as well as other paramyxoviruses. Viral genome encapsidated by N is as a template and recognized by RNA-dependent RNA polymerase complex composed of L and P. The offspring RNA also needs to assemble with N to form nucleocapsids. The N is one of the most abundant viral proteins in infected cells and chaperoned in the RNA-free form (N0) by P before encapsidation. In this study, we presented the structure of unassembled HPIV3 N0 in complex with the N-terminal portion of the P, revealing the molecular details of the N0 and the conserved N0-P interaction. Combined with biological experiments, we showed that the P binds to the C-terminal domain of N0 mainly by hydrophobic interaction and maintains the unassembled conformation of N by interfering with the formation of N-RNA oligomers, which might be a target for drug development. Based on the complex structure, we developed a method to obtain the monomeric N0. Furthermore, we designed a P-derived fusion peptide with 10-fold higher affinity, which hijacked the N and interfered with the binding of the N to RNA significantly. Finally, we proposed a model of conformational transition of N from the unassembled state to the assembled state, which helped to further understand viral replication. IMPORTANCE Human parainfluenza virus 3 (HPIV3) causes annual epidemics of respiratory diseases, especially in newborns and infants. For the replication of HPIV3 and other paramyxoviruses, only three viral proteins are required: phosphoprotein (P), RNA polymerase (L), and nucleoprotein (N). Here, we report the crystal structure of the complex of N and its chaperone P. We describe in detail how P acts as a chaperone to maintain the unassembled conformation of N. Our analysis indicated that the interaction between P and N is conserved and mediated by hydrophobicity, which can be used as a target for drug development. We obtained a high-affinity P-derived peptide inhibitor, specifically targeted N, and greatly interfered with the binding of the N to RNA, thereby inhibiting viral encapsidation and replication. In summary, our results provide new insights into the paramyxovirus genome replication and nucleocapsid assembly and lay the basis for drug development.

Structural Insights into the Ligand-Binding and -Releasing Mechanism of Helicoverpa armigera Pheromone-Binding Protein PBP1.

Cotton bollworm (Helicoverpa armigera) is a worldwide agricultural pest in which the transport of pheromones is indispensable and perceived by pheromone-binding proteins (PBPs). However, three-dimensional structure, pheromone binding, and releasing mechanisms of PBPs are not completely illustrated. Here, we solved three structures of the cotton bollworm HarmPBP1 at different pH values and its complex with ligand, Z-9-hexadecenal. Although apo-HarmPBP1 adopts a common PBP scaffold of six α-helices surrounding a predominantly hydrophobic central pocket, the conformation is greatly distinct from other apo-PBPs. The Z-9-hexadecenal is bound mainly by hydrophobic interaction. The pheromone can enter this cavity through an opening between the helices α5 and α6, as well as the loop between α3 and α4. Structural analysis suggests that ligand entry into the pocket is followed by a shift of Lys94 and Lys138, which may act as a lid at the opening of the pocket. Acidic pH will cause a subtle structural change of the lid, which in turn affects its ligand-binding ability, differently from other family proteins. Taken together, this study provides structural bases for the interactions between pheromones and PBPs, the pH-induced conformational switch, and the design of small inhibitors to control cotton bollworms by disrupting male-female chemosensory communication.

Using transfixion irrigation with negative pressure drainage (TINPD) minimally invasive to manage infratemporal fossa (ITF) abscess.

RATIONALE: The treatment of abscess in the infratemporal space is still controversial and bedside and operative intraoral drainage is often used to resolve the abscess. However, it can be difficult to control the infection quickly.[1] In this report, the authors present a new technique of using transfixion irrigation with negative pressure drainage for minimally invasive management of infratemporal fossa abscess. PATIENT CONCERNS: A 45-year-old man with type 2 diabetes complained of painful swelling and trismus in the right lower facial region for 10 days. The patient was weak, with mild anxiety, and gradually aggravated. DIAGNOSES: The patient was misdiagnosed and received dental pulp treatment for the right mandibular first molar and was given oral cefradine capsules (500 mg 3 times per day). Computed tomography scan and puncture revealed an abscess in the infratemporal fossa. INTERVENTION: The authors used transfixion irrigation with negative pressure drainage from different directions to reach the abscess cavity. Saline solution was infused through 1 tube and allowed to flow out through the other tube to flush out the pus and debris from the abscess. OUTCOME: On day 9, the drainage tube was removed and the patient was discharged. One week later, the patient was followed up in the outpatient clinic and the impacted mandibular third molar was removed. This technique is less invasive and leads to faster recovery times and fewer complications. LESSONS SUBSECTIONS: The report highlights the importance of proper preoperative evaluation, using a thoracic drainage tube as soon as possible, and continuous flushing. A double-lumen drainage tube with a suitable diameter and combined flushing should be designed for future reference. Moreover, the use of drugs can effectively eliminate emboli formation, allowing for faster and more minimally invasive control and removal of the infection.[2].

Structures and Functional Diversities of ASFV Proteins.

African swine fever virus (ASFV), the causative pathogen of the recent ASF epidemic, is a highly contagious double-stranded DNA virus. Its genome is in the range of 170~193 kbp and encodes 68 structural proteins and over 100 non-structural proteins. Its high pathogenicity strains cause nearly 100% mortality in swine. Consisting of four layers of protein shells and an inner genome, its structure is obviously more complicated than many other viruses, and its multi-layered structures play different kinds of roles in ASFV replication and survival. Each layer possesses many proteins, but very few of the proteins have been investigated at a structural level. Here, we concluded all the ASFV proteins whose structures were unveiled, and explained their functions from the view of structures. Those structures include ASFV AP endonuclease, dUTPases (E165R), pS273R protease, core shell proteins p15 and p35, non-structural proteins pA151R, pNP868R (RNA guanylyltransferase), major capsid protein p72 (gene B646L), Bcl-2-like protein A179L, histone-like protein pA104R, sulfhydryl oxidase pB119L, polymerase X and ligase. These novel structural features, diverse functions, and complex molecular mechanisms promote ASFV to escape the host immune system easily and make this large virus difficult to control.

Novel insights into the function of an N-terminal region of DENV2 NS4B for the optimal helicase activity of NS3.

Dengue virus NS3 is a prototypical DEx(H/D) helicase that binds and hydrolyzes NTP to translocate along and unwind double-stranded nucleic acids. NS3 and NS4B are essential components of the flavivirus replication complex. Evidences showed that NS4B interacted with NS3 and modulated the helicase activity of NS3. Despite important insights into structural, mechanistic, and cellular aspects of the NS3 function, there is still a gap in understanding how it coordinates the helicase activities within the replicase complex for efficient replication. Here, using the DENV2 as a model, we redefined the critical region of NS4B required for NS3 function by pull-down and MST assays. The FRET-based unwinding assay showed that NS3 would accelerate unwinding duplex nucleic acids in the presence of NS4B (51-83). The simulated NS3-NS4B complex models based on the rigid-body docking delineated the potential interaction sites located in the conserved motif within the core domain of NS3. Mutations in motif I (I190A) and motif III (P319L) of NS3 interfered with the unwinding activity stimulated by NS4B. Upon binding to the NS3 helicase, NS4B assisted NS3 to dissociate from single-stranded nucleic acid and enabled NS3 helicase to keep high activity at high ATP concentrations. These results suggest that NS4B probably serves as an essential cofactor for NS3 to coordinate the ATP cycles and nucleic acid binding during viral genome replication.

Long noncoding RNA and its role in virus infection and pathogenesis.

Long noncoding RNAs (lncRNAs) are RNA molecules longer than 200 nucleotides that regulate gene expression at the transcriptional and post-transcriptional levels. Emerging evidence showed that lncRNAs play important roles in a wide range of biological processes, including cell proliferation, apoptosis, and tumorigenesis. The infection of virus generally can regulate the expression of the cellular lncRNA expression. The lncRNAs which encoded by virus can also modulate the expression of the hosts' gene which is critical for virus infection. Here, we summarized the recent progress on long noncoding RNA and its relationship with virus, especially the function of long noncoding RNA on virus-induced oncogenesis. Studies on lncRNAs and their relationship with viruses may give new insights into virus-host interactions and therapy of related diseases.