Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases.

Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of the LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe that is not found in other OTU-DUBs. The structural topology of an extended helical lobe is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleave longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, whereas it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.

Integrative modeling of tumor genomes and epigenomes for enhanced cancer diagnosis by cell-free DNA.

Multi-cancer early detection remains a key challenge in cell-free DNA (cfDNA)-based liquid biopsy. Here, we perform cfDNA whole-genome sequencing to generate two test datasets covering 2125 patient samples of 9 cancer types and 1241 normal control samples, and also a reference dataset for background variant filtering based on 20,529 low-depth healthy samples. An external cfDNA dataset consisting of 208 cancer and 214 normal control samples is used for additional evaluation. Accuracy for cancer detection and tissue-of-origin localization is achieved using our algorithm, which incorporates cancer type-specific profiles of mutation distribution and chromatin organization in tumor tissues as model references. Our integrative model detects early-stage cancers, including those of pancreatic origin, with high sensitivity that is comparable to that of late-stage detection. Model interpretation reveals the contribution of cancer type-specific genomic and epigenomic features. Our methodologies may lay the groundwork for accurate cfDNA-based cancer diagnosis, especially at early stages.

Associations between local acidosis induced by renal LDHA and renal fibrosis and mitochondrial abnormalities in patients with diabetic kidney disease.

During the progression of diabetic kidney disease (DKD), renal lactate metabolism is rewired. The relationship between alterations in renal lactate metabolism and renal fibrosis in patients with diabetes has only been partially established due to a lack of biopsy tissues from patients with DKD and the intricate mechanism of lactate homeostasis. The role of lactate dehydrogenase A (LDHA)-mediated lactate generation in renal fibrosis and dysfunction in human and animal models of DKD was explored in this study. Measures of lactate metabolism (urinary lactate levels and LDHA expression) and measures of DKD progression (estimated glomerular filtration rate and Wilms' tumor-1 expression) were strongly negatively correlated in patients with DKD. Experiments with streptozotocin-induced DKD rat models and the rat renal mesangial cell model confirmed our findings. We found that the pathogenesis of DKD is linked to hypoxia-mediated lactic acidosis, which leads to fibrosis and mitochondrial abnormalities. The pathogenic characteristics of DKD were significantly reduced when aerobic glycolysis or LDHA expression was inhibited. Further studies will aim to investigate whether local acidosis caused by renal LDHA might be exploited as a therapeutic target in patients with DKD.

ClpL is a functionally active tetradecameric AAA+ chaperone, distinct from hexameric/dodecameric ones.

AAA+ (ATPases associated with diverse cellular activities) chaperones are involved in a plethora of cellular activities to ensure protein homeostasis. The function of AAA+ chaperones is mostly modulated by their hexameric/dodecameric quaternary structures. Here we report the structural and biochemical characterizations of a tetradecameric AAA+ chaperone, ClpL from Streptococcus pneumoniae. ClpL exists as a tetradecamer in solution in the presence of ATP. The cryo-EM structure of ClpL at 4.5 Å resolution reveals a striking tetradecameric arrangement. Solution structures of ClpL derived from small-angle X-ray scattering data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric AAA+ chaperone structures. Vertical positioning of the middle domain accounts for the head-to-head arrangement of two heptameric rings. Biochemical activity assays with site-directed mutagenesis confirmed the critical roles of residues both in the integrity of the tetradecameric arrangement and activities of ClpL. Non-conserved Q321 and R670 are crucial in the heptameric ring assembly of ClpL. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric AAA+ chaperones.

Neurogenin-1 Overexpression Increases the Therapeutic Effects of Mesenchymal Stem Cells through Enhanced Engraftment in an Ischemic Rat Brain.

BACKGROUND AND OBJECTIVES: Stem cell therapy is a promising strategy for treating neurological diseases but its effectiveness is influenced by the route of administration and the characteristics of the stem cells. We determined whether neural induction of mesenchymal stem cells (MSCs) was beneficial when the cells were delivered intra-arterially through the carotid artery. METHODS AND RESULTS: MSCs were neurally induced using a retroviral vector expressing the neurogenic transcription factor neurogenin-1 (Ngn1). The LacZ gene encoding bacterial ß-galactosidase was used as a control. Ischemic stroke was induced by transluminal occlusion of the middle cerebral artery and 3 days later the MSCs were delivered intra-arterially through the internal carotid artery. Magnetic resonance imaging analysis indicated that compared to MSCs expressing LacZ (MSCs/LacZ), MSCs expressing Ngn1 (MSCs/Ngn1) exhibited increased recruitment to the ischemic region and populated this area for a longer duration. Immunohistochemical analysis indicated that compared to MSCs/LacZ, MSCs/Ngn1 more effectively alleviated neurological dysfunction by blocking secondary damage associated with neuronal cell death and brain inflammation. Microarray and real-time PCR analysis indicated that MSCs/Ngn1 exhibited increased expression of chemotactic cytokine receptors, adherence to endothelial cells, and migration ability. CONCLUSIONS: Neural induction with Ngn1 increases the homing ability of MSCs, enhancing their engraftment efficiency in the ischemic rat brain. Intra-arterial delivery of neurally induced MSCs/Ngn1 3 days after ischemic injury blocks neuronal cell death and inflammation, and improves functional recovery. Thus, intra-arterial administration of stem cells with neural properties may be a novel therapy for the treatment of ischemic stroke.

Site-specific monoubiquitination downregulates Rab5 by disrupting effector binding and guanine nucleotide conversion.

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.

Electrical Detection of Pneumococcus through the Nanoparticle Decoration Method.

A simple method of nanoparticle decoration can be used in the detection of pneumococcus. After the pneumococcal bacteria were captured by an antibody (pneumococcal C-polysaccharide (PnC) antibody) between the interdigitated electrodes, the gold nanoparticles conjugated with the PnC antibodies were let to bind onto an outer membrane of the bacteria. Upon successfully dense decoration, the bacteria surface will become conductive owing to the metal nanoparticles, and a distinctive conductance change between the electrodes can be observed. Since this success ratio, or the probability of the conductance change, reflects the concentration of the analyte, a number of repeated measurements can be used in the quantification of the bacteria. In this way, we have successfully detected S. pneumoniae in the range of 10-108 CFU/mL. The limit of detection in this work is lower than that in the commercial detection kit. We hope that the nanoparticle decoration method will play a role in the facile detection of various bacteria.

IVAG: An Integrative Visualization Application for Various Types of Genomic Data Based on R-Shiny and the Docker Platform.

Next-generation sequencing (NGS) technology has become a trend in the genomics research area. There are many software programs and automated pipelines to analyze NGS data, which can ease the pain for traditional scientists who are not familiar with computer programming. However, downstream analyses, such as finding differentially expressed genes or visualizing linkage disequilibrium maps and genome-wide association study (GWAS) data, still remain a challenge. Here, we introduce a dockerized web application written in R using the Shiny platform to visualize pre-analyzed RNA sequencing and GWAS data. In addition, we have integrated a genome browser based on the JBrowse platform and an automated intermediate parsing process required for custom track construction, so that users can easily build and navigate their personal genome tracks with in-house datasets. This application will help scientists perform series of downstream analyses and obtain a more integrative understanding about various types of genomic data by interactively visualizing them with customizable options.

Molecular characterization of a single-chain antibody variable fragment (scFv) specific for PspA from Streptococcus pneumoniae.

Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.

Comparison of MSC-Neurogenin1 administration modality in MCAO rat model.

Intracerebral (IC) grafting of mesenchymal stem cells (MSCs) is not currently used in humans due to its potential complications. On the other hand, intra-arterial (IA) administration can be facilitated for engrafting of intensifed MSCs in the injured human brain. The study is designed to compare the two methods of MSC administration using IA and IC routes through the parameters of behavior, infarct volume, cell distribution, and MSC identification. An ischemic stroke model was generated in Sprague Dawley male rats. This experiment used MSCs/Ngn1 that express Neurogenin1 (Ngn1) to ensure grafted MSC maintenance. MSCs/Ngn1 or normal saline was administrated via the IC or IA route on day 3. All animals were randomly assigned into four groups (five rats in each group): IC-control, IA-control, IC-MSCs/Ngn1, or IA-MSCs/Ngn1. Motor behaviors, infarct volume, and distribution of superparamagnetic iron oxide (SPIO)-labeled cells on magnetic resonance imaging (MRI) were compared from each group. There were no baseline differencess in motor behaviors or infarct volume between IC-MSCs/Ngn1 and IA-MSCs/Ngn1. Hovever, the IA-MSCs/Ngn1 group showed the greatest recovery on Rotarod testing and adhesive removal tests (p = 0.003 and p = 0.009 vs. IC-MSCs/Ngn1, respectively). The IA-MSCs/Ngn1 group also had more evenly distributed SPIO-labeled cells on MRI. The results suggest that IA administration is likely to be benefcial for humans based on its ability to improve behavioral outcomes and ensure even MSC engrafting.

Molecular determinants of polyubiquitin recognition by continuous ubiquitin-binding domains of Rad18.

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub2-Ub4) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the "extended LR motif" (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub2, not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.

Structural and biochemical characterizations of an intramolecular tandem coiled coil protein.

Coiled coil has served as an excellent model system for studying protein folding and developing protein-based biomaterials. Most designed coiled coils function as oligomers, namely intermolecular coiled coils. However, less is known about structural and biochemical behavior of intramolecular coiled coils where coiled coil domains are covalently linked in one polypeptide. Here we prepare a protein which harbors three coiled coil domains with two short linkers, termed intramolecular tandem coiled coil (ITCC) and characterize its structural and biochemical behavior in solution. ITCC consists of three coiled coil domains whose sequences are derived from Coil-Ser and its domain swapped dimer. Modifications include positioning E (Glu) residue at "e" and K (Lys) at "g" positions throughout heptad repeats to enhance ionic interaction among its constituent coiled coil domains. Molecular modeling of ITCC suggests a compact triple helical bundle structure with the second and the third coiled coil domains forming a canonical coiled coil. ITCC exists as a mixture of monomeric and dimeric species in solution. Small-angle X-ray scattering reveals ellipsoidal molecular envelopes for both dimeric and monomeric ITCC in solution. The theoretically modeled structures of ITCC dock well into the envelopes of both species. Higher ionic strength shifts the equilibrium into monomer with apparently more compact structure while secondary structure remains unchanged. Taken together, our results suggest that our designed ITCC is predominantly monomeric structure through the enhanced ionic interactions, and its conformation is affected by the concentration of ionic species in the buffer.

Immune following suppression mesenchymal stem cell transplantation in the ischemic brain is mediated by TGF-ß.

Transplantation of mesenchymal stem cells (MSCs) has been shown to enhance the recovery of brain functions following ischemic injury. Although immune modulation has been suggested to be one of the mechanisms, the molecular mechanisms underlying improved recovery has not been clearly identified. Here, we report that MSCs secrete transforming growth factor-beta (TGF-ß) to suppress immune propagation in the ischemic rat brain. Ischemic stroke caused global death of resident cells in the infarcted area, elevated the monocyte chemoattractant protein-1 (MCP-1) level, and evoked massive infiltration of circulating CD68+ immune cells through the impaired blood-brain barrier. Transplantation of MSCs at day 3 post-ischemia blocked the subsequent upregulation of MCP-1 in the ischemic area and the infiltration of additional CD68+ immune cells. MSC-conditioned media decreased the migration and MCP-1 production of freshly isolated immune cells in vitro, and this effect was blocked by an inhibitor of TGF-ß signaling or an anti-TGF-ß neutralizing antibody. Finally, transplantation of TGF-ß1-silenced MSCs failed to attenuate the infiltration of CD68+ cells into the ischemic brain, and was associated with only minor improvements in motor function. These results indicate that TGF-ß is key to the ability of MSCs to beneficially attenuate immune reactions in the ischemic brain. Our findings offer insight into the interactions between allogeneic MSCs and the host immune system, reinforcing the prospective clinical value of using MSCs in the treatment of neurological disorders involving inflammation-mediated secondary damage.