Structural basis for promoter DNA recognition by the response regulator OmpR.

OmpR, a response regulator of the EnvZ/OmpR two-component system (TCS), controls the reciprocal regulation of two porin proteins, OmpF and OmpC, in bacteria. During signal transduction, OmpR (OmpR-FL) undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (OmpRn) and recognizes the promoter DNA from its C-terminal DNA-binding domain (OmpRc) to elicit an adaptive response. Apart from that, OmpR regulates many genes in Escherichia coli and is important for virulence in several pathogens. However, the molecular mechanism of the regulation and the structural basis of OmpR-DNA binding is still not fully clear. In this study, we presented the crystal structure of OmpRc in complex with the F1 region of the ompF promoter DNA from E. coli. Our structural analysis suggested that OmpRc binds to its cognate DNA as a homodimer, only in a head-to-tail orientation. Also, the OmpRc apo-form showed a unique domain-swapped crystal structure under different crystallization conditions. Biophysical experimental data, such as NMR, fluorescent polarization and thermal stability, showed that inactive OmpR-FL (unphosphorylated) could bind to promoter DNA with a weaker binding affinity as compared with active OmpR-FL (phosphorylated) or OmpRc, and also confirmed that phosphorylation may only enhance DNA binding. Furthermore, the dimerization interfaces in the OmpRc-DNA complex structure identified in this study provide an opportunity to understand the regulatory role of OmpR and explore the potential for this "druggable" target.

Direct Antibody Isolation on Cells Using Affinity-Tag-Guided Proximity Selection.

Traditional antibody generation, using either phage display or animal immunization, relies on purified antigens. Many membrane proteins, such as G protein-coupled receptors, solute carriers, or ion channels, are important drug targets but very challenging for the formation of antibodies due to the difficulty of protein purification. Whole-cell panning is an alternative approach for generating antibodies without the need for antigen purification. However, it often suffers from background interference and therefore requires extensive screening with low success rates. Here, we develop a new phage selection method, dubbed affinity-tag-guided proximity selection (A-GPS), to efficiently isolate specific antibodies directly from the antigen-presenting cells. By engineering a genetically fused affinity tag for the target antigen, A-GPS confines the proximity labeling reaction near the target antigen and preferentially enriches the phage bound to the target antigen. Using surface-presented GFP on human cells as a model antigen, we demonstrated that A-GPS successfully enriched the antigen-specific clones in two rounds of selection. Among the 46 randomly picked clones, >95% of clones showed great affinity and specificity for GFP over the background of HEK293T surface proteins. One of the best clones expressed as a Fab fragment showed subnanomolar binding affinity for GFP. This clone was successfully applied to common biological applications, such as immunofluorescence and flow cytometry, reflecting the usefulness of A-GPS for generating commercial-grade antibodies.

Establishment of a patient-derived organoid model and living biobank for nasopharyngeal carcinoma.

Background: Recurrent nasopharyngeal carcinoma (NPC) remains a major challenge for clinicians and scientists. Tumor organoid is a revelational disease model that highly resembled the heterogeneity and histopathological characteristics of original tumors. This study aimed to optimize the modeling process of patient-derived NPC organoids (NPCOs), and establish a living-biobank of NPCs to study the mechanism and explore the more effective treatment of the disease. Methods: Sixty-two fresh NPC tissue samples and 15 normal mucosa samples were collected for 3-dimensional (3D) organoid culture. The organoids were confirmed using hematoxylin and eosin assays. The expression levels of CD133, CD44, BMI-1, and Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were detected by immunohistochemistry (IHC) and in situ hybridization (ISH). Recurrent NPCOs were frozen in liquid nitrogen for 6 months and then resuscitated and passaged. Results: We identified a novel two-step enzymatic strategy for the treatment of NPC and nasal mucosa specimens and an optimal medium for culturing NPCOs and nasal mucosa organoids (NMOs). Organoid cultures were generated from 34 primary NPC samples, 28 recurrent NPC samples, and 15 normal mucosa samples. The success rates for primary NPCO, recurrent NPCO, and NMO formation were 47.06%, 81.25%, and 86.5%, respectively. All the NPCOs were EBER positive and CK7 negative. Recurrent NPCOs had higher expressions of stem cell markers, including BMI-1, CD44, and CD133. Additionally, recurrent NPCOs could be cultured to passage 4 and frozen and revived repeatedly, while primary NPCOs were challenging to culture. Conclusions: In summary, we successfully established a living biobank using the NPCO model, which has enormous potential in basic and clinical research on NPC.

Structural insights into DNA binding domain of vancomycin-resistance-associated response regulator in complex with its promoter DNA from Staphylococcus aureus.

In Staphylococcus aureus, vancomycin-resistance-associated response regulator (VraR) is a part of the VraSR two-component system, which is responsible for activating a cell wall-stress stimulon in response to an antibiotic that inhibits cell wall formation. Two VraR-binding sites have been identified: R1 and R2 in the vraSR operon control region. However, the binding of VraR to a promoter DNA enhancing downstream gene expression remains unclear. VraR contains a conserved N-terminal receiver domain (VraRN ) connected to a C-terminal DNA binding domain (VraRC ) with a flexible linker. Here, we present the crystal structure of VraRC alone and in complex with R1-DNA in 1.87- and 2.0-Å resolution, respectively. VraRC consisting of four α-helices forms a dimer when interacting with R1-DNA. In the VraRC -DNA complex structure, Mg2+ ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg2+ to VraRC enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraRC with R1- and R2-DNA revealed the essential residues that might play a crucial role in interacting with DNA of the vraSR operon. The structural information could help in designing and screening potential therapeutics/inhibitors to deal with antibiotic-resistant S. aureus via targeting VraR.

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method.

Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

Turning the RING domain protein MdmX into an active ubiquitin-protein ligase.

The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, although Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX, and we identified two distinct regions that, when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the cryptic nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but also acts as an allosteric activator of UbcH5b.

miR-223-3p Inhibits Antigen Endocytosis and Presentation and Promotes the Tolerogenic Potential of Dendritic Cells through Targeting Mannose Receptor Signaling and Rhob.

BACKGROUND: The role of miR-223-3p in dendritic cells (DCs) is unknown. This study is aimed at investigating the effect of miR-223-3p on the antigen uptake and presentation capacities of DCs and the underlying molecular mechanism. METHODS: FITC-OVA antigen uptake and cell surface markers in bone marrow-derived DCs (BMDCs) were analyzed by flow cytometry. BMDCs were transfected with the miR-223-3p mimic or inhibitor. Cytokine levels were determined by ELISA. CD4+ T cell differentiation was determined by mixed lymphocyte culture assay. RESULTS: OVA treatment significantly downregulated miR-223-3p in BMDCs. The miR-223-3p mimic significantly inhibited OVA-induced antigen uptake and surface expression of MHC-II on BMDCs (P < 0.01). The miR-223-3p mimic increased TGF-ß1 production in OVA-treated DCs (P < 0.01). Mixed lymphocyte reaction showed that the miR-223-3p mimic significantly promoted Treg cell differentiation. In addition, the miR-223-3p mimic significantly upregulated CD103 in DCs, indicating the promotion of tolerogenic DCs. The miR-223-3p mimic downregulated Rhob protein in OVA-induced DCs. Rhob knockdown significantly suppressed the ability of FITC-OVA endocytosis (P < 0.01) and surface MHC-II molecule expression (P < 0.01) in BMDCs, promoting promoted Treg cell differentiation. Mannose receptor (MR) knockdown significantly upregulated miR-223-3p, downregulated Rhob protein in OVA-treated DCs, inhibited the FITC-OVA endocytosis and surface MHC-II expression in BMDCs, and promoted Treg cell differentiation (all P < 0.01). CONCLUSION: These data suggest that miR-223-3p has an inhibitory effect on the antigen uptake and presentation capacities of BMDCs and promotes Treg cell differentiation, which is, at least partially, through targeting MR signaling and Rhob.