Constructing training set using distance between learnt graphical models of time series data on patient physiology, to predict disease scores.

Interventional endeavours in medicine include prediction of a score that parametrises a new subject's susceptibility to a given disease, at the pre-onset stage. Here, for the first time, we provide reliable learning of such a score in the context of the potentially-terminal disease VOD, that often arises after bone marrow transplants. Indeed, the probability of surviving VOD, is correlated with early intervention. In our work, the VOD-score of each patient in a retrospective cohort, is defined as the distance between the (posterior) probability of a random graph variable-given the inter-variable partial correlation matrix of the time series data on variables that represent different aspects of patient physiology-and that given such time series data of an arbitrarily-selected reference patient. Such time series data is recorded from a pre-transplant to a post-transplant time, for each patient in this cohort, though the data available for distinct patients bear differential temporal coverage, owing to differential patient longevities. Each graph is a Soft Random Geometric Graph drawn in a probabilistic metric space, and the computed inter-graph distance is oblivious to the length of the time series data. The VOD-score learnt in this way, and the corresponding pre-transplant parameter vector of each patient in this retrospective cohort, then results in the training data, using which we learn the function that takes VOD-score as its input, and outputs the vector of pre-transplant parameters. We model this function with a vector-variate Gaussian Process, the covariance structure of which is kernel parametrised. Such modelling is easier than if the score variable were the output. Then for any prospective patient, whose pre-transplant variables are known, we learn the VOD-score (and the hyperparameters of the covariance kernel), using Markov Chain Monte Carlo based inference.

Reconditioned monocytes are immunomodulatory and regulate inflammatory environment in sepsis.

Sepsis is caused by dysregulated immune response to severe infection and hyper inflammation plays a central role in worsening the disease. The immunomodulatory properties of mesenchymal stem cells (MSCs) have been evaluated as a therapeutic candidate for sepsis. Reconditioned monocytes (RM), generated from healthy human peripheral blood mononuclear cells (PBMCs) exhibit both macrophage and MSCs-like properties. RM were administered at different stages of sepsis in a mouse model. It reduced serum levels of IL6, MCP-1, IL-10, improved hypothermia, increased survival, and recovery from 0 to 66% when combined with antibiotics in the mouse model. The reduced human leucocyte antigen DR molecules expression on RM enables their co-culture with PBMCs of sepsis patients which resulted in reduced ROS production, and up-regulated TGF-ß while down-regulating IL6, IL8, and IL-10 in-vitro. RM are potentially immunomodulatory, enhance survival in sepsis mouse model and modulate inflammatory behaviour of sepsis patient's PBMCs.

Type 1 interferon mediated signaling is indispensable for eliciting anti-tumor responses by Mycobacterium indicus pranii.

Introduction: The evolving tumor secretes various immunosuppressive factors that reprogram the tumor microenvironment (TME) to become immunologically cold. Consequently, various immunosuppressive cells like Tregs are recruited into the TME which in turn subverts the anti-tumor response of dendritic cells and T cells.Tumor immunotherapy is a popular means to rejuvenate the immunologically cold TME into hot. Mycobacterium indicus pranii (MIP) has shown strong immunomodulatory activity in different animal and human tumor models and has been approved for treatment of lung cancer (NSCLC) patients as an adjunct therapy. Previously, MIP has shown TLR2/9 mediated activation of antigen presenting cells/Th1 cells and their enhanced infiltration in mouse melanoma but the underlying mechanism by which it is modulating these immune cells is not yet known. Results: This study reports for the first time that MIP immunotherapy involves type 1 interferon (IFN) signaling as one of the major signaling pathways to mediate the antitumor responses. Further, it was observed that MIP therapy significantly influenced frequency and activation of different subsets of T cells like regulatory T cells (Tregs) and CD8+ T cells in the TME. It reduces the migration of Tregs into the TME by suppressing the expression of CCL22, a Treg recruiting chemokine on DCs and this process is dependent on type 1 IFN. Simultaneously, in a type 1 IFN dependent pathway, it enhances the activation and effector function of the immunosuppressive tumor resident DCs which in turn effectively induce the proliferation and effector function of the CD8+ T cells. Conclusion: This study also provides evidence that MIP induced pro-inflammatory responses including induction of effector function of conventional dendritic cells and CD8+ T cells along with reduction of intratumoral Treg frequency are essentially mediated in a type 1 IFN-dependent pathway.

Mycobacterium indicus pranii therapy suppresses systemic dissemination of tumor cells in B16F10 murine model of melanoma.

Cancer associated morbidity is mostly attributed to the dissemination of tumor cells from their primary niche into the circulation known as "metastasis". Mycobacterium indicus pranii (MIP) an approved immunotherapeutic agent against lung cancer (NSCLC) has shown potent anti-tumor activity in prior studies. While evaluating anti-tumor activity of MIP in mouse model, MIP treated animals typically exhibited less metastatic lesions in their pulmonary compartment. To study the role of MIP in metastasis closely, B16F10 melanoma cells were implanted subcutaneously in the mice, and the dissemination of tumor cells from the solid tumor was evaluated over a period of time. When B16F10 melanoma cells were treated with MIP in vitro, downregulation of epithelial mesenchymal transition markers was observed in these cells, which in turn suppressed the invasion, migration and adhesion of tumor cells. Notably, MIP therapy was found to be effectively reducing the metastatic burden in murine model of melanoma. Molecular characterization of MIP treated tumor cells substantiated that MIP upregulates the PPARγ expression within the tumor cells, which attenuates the NFκB/p65 levels within the nucleus, resulting in the suppression of Mmp9 expression in tumor cells. Besides that, MIP also downregulated the surface expression of chemokine receptor CXCR4 in murine melanoma cells, where chromatin immunoprecipitation confirmed the impeded recruitment of p50 and c-Rel factors to the Cxcr4 promoter, resulting in its downregulation transcriptionally. Taken together, MIP suppressed the dissemination of tumor cells in vivo, by regulating the expression of MMP9 and CXCR4 on these cells.

Tumor targeted delivery of mycobacterial adjuvant encapsulated chitosan nanoparticles showed potential anti-cancer activity and immune cell activation in tumor microenvironment.

Targeting immunotherapeutics inside the tumor microenvironment (TME) with intact biological activity remains a pressing issue. Mycobacterium indicus pranii (MIP), an approved adjuvant therapy for leprosy has exhibited promising results in clinical trials of lung (NSCLC) and bladder cancer. Whole MIP as well as its cell wall fraction have shown tumor growth suppression and enhanced survival in mice model of melanoma, when administered peritumorally. Clinically, peritumoral delivery remains a procedural limitation. In this study, a tumor targeted delivery system was designed, where chitosan nanoparticles loaded with MIP adjuvants, when administered intravenously showed preferential accumulation within the TME, exploiting the principle of enhanced permeability and retention effect. Bio-distribution studies revealed their highest concentration inside the tumor after 6 h of administration. Interestingly, MIP adjuvant nano-formulations significantly reduced the tumor volume in the treated groups and increased the frequency of activated immune cells inside the TME. For chemoimmunotherapeutics studies, MIP nano-formulation was combined with standard dosage regimen of Paclitaxel. Combined therapy exhibited a further reduction in tumor volume relative to either of the MIP nano formulations. From this study a three-pronged strategy emerged as the underlying mechanism; chitosan and Paclitaxel have shown direct role in tumor cell death and the MIP nano-formulation activates the tumor residing immune cells which ultimately leads to the reduced tumor growth.

Chromatin-Remodeling Factor BRG1 Is a Negative Modulator of L. donovani in IFNγ Stimulated and Infected THP-1 Cells.

Intracellular pathogens manipulate the host cell for their own survival by contributing to modifications of host epigenome, and thus, altering expression of genes involved in the pathogenesis. Both ATP-dependent chromatin remodeling complex and histone modifications has been shown to be involved in the activation of IFNγ responsive genes. Leishmania donovani is an intracellular pathogen that causes visceral leishmaniasis. The strategies employed by Leishmania donovani to modulate the host epigenome in order to overcome the host defense for their persistence has been worked out in this study. We show that L. donovani negatively affects BRG1, a catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, to alter IFNγ induced host responses. We observed that L. donovani infection downregulates BRG1 expression both at transcript and protein levels in cells stimulated with IFNγ. We also observed a significant decrease in IFNγ responsive gene, Class II transactivator (CIITA), as well as its downstream genes, MHC-II (HLA-DR and HLA-DM). Also, the occupancy of BRG1 at CIITA promoters I and IV was disrupted. A reversal in CIITA expression and decreased parasite load was observed with BRG1 overexpression, thus, suggesting BRG1 is a potential negative regulator for the survival of intracellular parasites in an early phase of infection. We also observed a decrease in H3 acetylation at the promoters of CIITA, post parasite infection. Silencing of HDAC1, resulted in increased CIITA expression, and further decreased parasite load. Taken together, we suggest that intracellular parasites in an early phase of infection negatively regulates BRG1 by using host HDAC1 for its survival inside the host.

Macrophages Are the Key Players in Promoting Hyper-Inflammatory Response in a Mouse Model of TB-IRIS.

TB-IRIS is an abnormal inflammatory response in a subset of HIV-TB co-infected patients shortly after initiation of anti-retroviral therapy (ART). Therapy in these patients could have greatly improved the life expectancy as ART reconstitutes the function and number of CD4+ T cells and many patients see improvement in symptoms but paradoxically up to 54% of co-infected patients develop TB-IRIS. Different studies have indicated that both innate and adaptive immunity are involved in the pathology of IRIS but the role of macrophages in abnormal activation of CD4+ T cells is poorly understood. Since macrophages are one of the major antigen-presenting cells and are infected by M.tb at a high frequency, they are very much likely to be involved in the development of TB-IRIS. In this study, we have developed a mouse model of experimental IRIS, in which M.tb-infected T-cell knockout mice undergo a fatal inflammatory disease after CD4+ T cell reconstitution. Lung macrophages and blood monocytes from M.tb-infected TCRß-/- mice showed upregulated expression of cell surface activation markers and also showed higher mRNA expression of inflammation-associated chemokines and matrix metalloproteases responsible for tissue damage. Furthermore, cytokine and TLR signaling feedback mechanism to control excessive inflammation was also found to be dysregulated in these macrophages under lymphopenic conditions. Previous studies have shown that hyperactive CD4+ T cells are responsible for disease induction and our study shows that somehow macrophages are in a higher activated state when infected with M.tb in an immune-deficient condition, which results in excessive activation of the adoptively transferred CD4+ T cells. Understanding of the mechanisms underlying the pathophysiology of TB-IRIS would facilitate identification of prospective biomarkers for disease development in HIV-TB co-infected patients before starting antiretroviral therapy.

Epigenetic regulation of defense genes by histone deacetylase1 in human cell line-derived macrophages promotes intracellular survival of Leishmania donovani.

Leishmania donovani, an intracellular protozoan parasite upon infection, encounters a range of antimicrobial factors within the host cells. Consequently, the parasite has evolved mechanisms to evade this hostile defense system through inhibition of macrophage activation that, in turn, enables parasite replication and survival. There is growing evidence that epigenetic down-regulation of the host genome by intracellular pathogens leads to acute infection. Epigenetic modification is mediated by chromatin remodeling, histone modifications, or DNA methylation. Histone deacetylases (HDACs) removes acetyl groups from lysine residues on histones, thereby leading to chromatin remodeling and gene silencing. Here, using L. donovani infected macrophages differentiated from THP-1 human monocytic cells, we report a link between host chromatin modifications, transcription of defense genes and intracellular infection with L. donovani. Infection with L. donovani led to the silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) transcript levels, protein expression, and enzyme activity showed a significant increase upon infection. HDAC1 occupancy at the promoters of the defense genes significantly increased upon infection, which in turn resulted in decreased histone H3 acetylation in infected cells, resulting in the down-regulation of mRNA expression of host defense genes. Small molecule mediated inhibition and siRNA mediated down-regulation of HDAC1 increased the expression levels of host defense genes. Interestingly, in this study, we demonstrate that the silencing of HDAC1 by both siRNA and pharmacological inhibitors resulted in decreased intracellular parasite survival. The present data not only demonstrate that up-regulation of HDAC1 and epigenetic silencing of host cell defense genes is essential for L. donovani infection but also provides novel therapeutic strategies against leishmaniasis.

Development of a high yielding expression platform for the introduction of non-natural amino acids in protein sequences.

The ability to genetically encode non-natural amino acids (nnAAs) into proteins offers an expanded tool set for protein engineering. nnAAs containing unique functional moieties have enabled the study of post-translational modifications, protein interactions, and protein folding. In addition, nnAAs have been developed that enable a variety of biorthogonal conjugation chemistries that allow precise and efficient protein conjugations. These are being studied to create the next generation of antibody-drug conjugates with improved efficacy, potency, and stability for the treatment of cancer. However, the efficiency of nnAA incorporation, and the productive yields of cell-based expression systems, have limited the utility and widespread use of this technology. We developed a process to isolate stable cell lines expressing a pyrrolysyl-tRNA synthetase/tRNApyl pair capable of efficient nnAA incorporation. Two different platform cell lines generated by these methods were used to produce IgG-expressing cell lines with normalized antibody titers of 3 g/L using continuous perfusion. We show that the antibodies produced by these platform cells contain the nnAA functionality that enables facile conjugations. Characterization of these highly active and robust platform hosts identified key parameters that affect nnAA incorporation efficiency. These highly efficient host platforms may help overcome the expression challenges that have impeded the developability of this technology for manufacturing proteins with nnAAs and represents an important step in expanding its utility.

qPCR assays to quantitate tRNApyl and pylRS expression in engineered cell lines.

Non-natural amino acids (nnAA) contain unique functional moieties that greatly expand the available tool set for protein engineering. But incorporation of nnAAs requires the function of an orthogonal aminoacyl tRNA synthetase/tRNA pair. Stable cell lines expressing these components have been shown capable of producing gram per liter levels of antibodies with nnAAs. However, little has been reported on the genetic makeup of these cells. To gain a better understanding of the minimal requirements for efficient nnAA incorporation we developed qPCR methods for the quantitation of the key components. Here we describe the development of qPCR assays for the quantification of tRNApyl and pylRS. qPCR was chosen because it provides a large dynamic range, has high specificity for its target, and is a non-radioactive method used routinely for cell line characterization. Designing assays for tRNAs present challenges due to their short length (~72 nucleotides) and high secondary structure. These tRNA assays have a ≥ 5 log dynamic range with the tRNApyl assays being able to discern the mature and unprocessed forms of the tRNApyl. Cell line analysis showed tRNApyl was expressed at higher levels than the CHO-K1 endogenous Met and Phe tRNAs and that >88% of tRNApyl was the mature form.

Amber suppression coupled with inducible surface display identifies cells with high recombinant protein productivity.

Cell line development (CLD) for biotherapeutics is a time- and resource-intensive process requiring the isolation and screening of large numbers of clones to identify high producers. Novel methods aimed at enhancing cell line screening efficiency using markers predictive of productivity early in the CLD process are needed to reliably generate high-yielding cell lines. To enable efficient and selective isolation of antibody expressing Chinese hamster ovary cells by fluorescence-activated cell sorting, we developed a strategy for the expression of antibodies containing a switchable membrane-associated domain to anchor an antibody to the membrane of the expressing cell. The switchable nature of the membrane domain is governed by the function of an orthogonal aminoacyl transfer RNA synthetase/tRNApyl pair, which directs a nonnatural amino acid (nnAA) to an amber codon encoded between the antibody and the membrane anchor. The process is "switchable" in response to nnAA in the medium, enabling a rapid transition between the surface display and secretion. We demonstrate that the level of cell surface display correlates with productivity and provides a method for enriching phenotypically stable high-producer cells. The strategy provides a means for selecting high-producing cells with potential applications to multiple biotherapeutic protein formats.

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies.

The conserved glycosylation site Asn297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment, important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality.

Sequential screening by ClonePix FL and intracellular staining facilitate isolation of high producer cell lines for monoclonal antibody manufacturing.

Screening and characterization of cell lines for stable production are critical tasks in identifying suitable recombinant cell lines for the manufacture of protein therapeutics. To aid this essential function we have developed a methodology for the selection of antibody expressing cells using fluorescence based ClonePix FL colony isolation and flow cytometry analysis following intracellular staining for immunoglobulin G (IgG). Our data show that characterization of cells by flow cytometry early in the clone selection process enables the identification of cell lines with the potential for high productivity and helps to eliminate unstable cell lines. We further demonstrate a correlation between specific productivity (qP) and intracellular heavy chain (HC) content with final productivity. The unique combination of screening using ClonePix FL and the flow cytometry approaches facilitated more efficient isolation of clonal cell lines with high productivity within a 15week timeline and which can be applied across NS0 and CHO host platforms. Furthermore, in this study we describe the critical parameters for the ClonePix FL colony based selection and the associated calculations to provide an assessment of the probability of monoclonality of the resulting cell lines.

Development of a fluorescent reporter system for monitoring ER stress in Chinese hamster ovary cells and its application for therapeutic protein production.

Mammalian cell expression systems have become a workhorse for the production of biotherapeutic proteins. As such, there is an ever increasing demand for higher productivity from these expression platforms to reduce manufacturing costs. While great advances have been made in the optimization of culture conditions and cell line selection to improve productivity, protein mis-folding remains a common limitation to high levels of production of therapeutic proteins. Accumulation of mis- and unfolded protein in the endoplasmic reticulum (ER) causes ER stress and initiates the unfolded protein response (UPR) that results in an activation of protein folding machinery, translation attenuation in an effort to proper folding of the newly synthesized peptides or may even lead to apoptosis if the correct folding is not restored. As a result, UPR associated apoptosis often results in lower protein expression. To better understand the molecular mechanisms in these pathways, we developed a reporter construct that detects Inositol-requiring enzyme 1 (IRE1)-alpha mediated splicing of X-box binding protein 1 (XBP1) to monitor the course of UPR activation in cell lines expressing monoclonal antibodies. Using this reporter we observed a clear activation of UPR in cells treated with known ER stress causing pharmacological agents, such as Tunicamycin (Tm) and Thapsigargin (Tg), as well as in stable IgG expressing cells during fed-batch cultures. Furthermore, we developed a stress metric that we term as ER stress index (ERSI) to gauge basal ER stress in cells which we used as a predictive tool for isolation of high IgG expressing cell lines. This reporter system, with its ability to monitor the stress involved in recombinant protein expression, has utility to assist in devising engineering strategies for improved production of biotherapeutic drugs.

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels.

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.

A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease.

Mammalian apurinic/apyrimidinic endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are highly toxic, mutagenic, and implicated in carcinogenesis. Also, reducing the activity of APE1 protein in cancer cells and tumors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. In general, mouse models are used in studies of basic mechanisms of carcinogenesis, as well as pre-clinical studies before transitioning into humans. Human APE1 (hAPE1) has previously been cloned, expressed, and extensively characterized. However, the knowledge regarding the characterization of mouse APE1 (mAPE1) is very limited. Here we have expressed and purified full-length hAPE1 and mAPE1 in and from E. coli to near homogeneity. mAPE1 showed comparable fast reaction kinetics to its human counterpart. Steady-state enzyme kinetics showed an apparent K(m) of 91 nM and k(cat) of 4.2 s(-1) of mAPE1 for the THF cleavage reaction. For hAPE1 apparent K(m) and k(cat) were 82 nM and 3.2 s(-1), respectively, under similar reaction conditions. However, k(cat)/K(m) were in similar range for both APE1s. The optimum pH was in the range of 7.5-8 for both APE1s and had an optimal activity at 50-100 mM KCl, and they showed Mg(2+) dependence and abrogation of activity at high salt. Circular dichroism spectroscopy revealed that increasing the Mg(2+) concentration altered the ratio of "turns" to "ß-strands" for both proteins, and this change may be associated with the conformational changes required to achieve an active state. Overall, compared to hAPE1, mAPE1 has higher K(m) and k(cat) values. However, overall results from this study suggest that human and mouse APE1s have mostly similar biochemical and biophysical properties. Thus, the conclusions of mouse studies to elucidate APE1 biology and its role in carcinogenesis may be extrapolated to apply to human biology. This includes the development and validation of effective APE1 inhibitors as chemosensitizers in clinical studies.

Interplay between ER exit code and domain conformation in CFTR misprocessing and rescue.

Multiple mutations in cystic fibrosis transmembrane conductance regulator (CFTR) impair its exit from the endoplasmic reticulum (ER). We compared two processing mutants: DeltaF508 and the ER exit code mutant DAA. Although both have severe kinetic processing defect, DAA but not DeltaF508 has substantial accumulation in its mature form, leading to higher level of processing at the steady state. DAA has much less profound conformational abnormalities. It has lower Hsp70 association and higher post-ER stability than DeltaF508. The ER exit code is necessary for DeltaF508 residual export and rescue. R555K, a mutation that rescues DeltaF508 misprocessing, improves Sec24 association and enhances its post-ER stability. Using in situ limited proteolysis, we demonstrated a clear change in trypsin sensitivity in DeltaF508 NBD1, which is reversed, together with that of other domains, by low temperature, R555K or both. We observed a conversion of the proteolytic pattern of DAA from the one resembling DeltaF508 to the one similar to wild-type CFTR during its maturation. Low temperature and R555K are additive in improving DeltaF508 conformational maturation and processing. Our data reveal a dual contribution of ER exit code and domain conformation to CFTR misprocessing and underscore the importance of conformational repair in effective rescue of DeltaF508.

Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, induced by alkylating chemotherapeutics, and deaminated and lipid peroxidation-induced purine adducts. We have generated monoclonal antibodies (moAbs) against human MPG. Twelve independent hybridoma clones were characterized, which, except 520-16A, are identical based on epitope exclusion assay. Four moAbs, including 520-2A, 520-3A, 520-16A, and 520-26A, have high affinity (K(D) approximately 0.3-1.6nM), and their subtypes were IgG(2a), IgG(1), IgG(2a), and IgG(2b), respectively. moAb 520-3A recognizes the sequence (52)AQAPCPRERCLGPP(66)T, an epitope exclusively present in the N-terminal extension of human MPG. We found that moAb 520-3A significantly inhibited MPG's enzymatic activity towards different substrates, such as hypoxanthine, 1,N(6)ethenoadenine and methylated bases, which represent different classes of DNA damage, however, with different efficiencies. Real-time binding experiments using surface plasmon resonance (SPR) spectroscopy showed that the pronounced inhibition of activity was not in the substrate-binding step. Single turnover kinetics (STO) revealed that the inhibition was at the catalytic step. Since we found that this antibody has an epitope in the N-terminal tail, the latter appears to have an important role in substrate discrimination, however, with a differential effect on different substrates.

Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage.

The DNA base excision repair (BER) is a ubiquitous mechanism for removing damage from the genome induced by spontaneous chemical reaction, reactive oxygen species (ROS) and also DNA damage induced by a variety of environmental genotoxicants. DNA repair is essential for maintaining genomic integrity. As we learn more about BER, a more complex mechanism emerges which supersedes the classical, simple pathway requiring only four enzymatic reactions. The key to understand the complete BER process is to elucidate how multiple proteins interact with one another in a coordinated process under specific physiological conditions.