New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.     

High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses

A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R² = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.     

Patterns of Human Papillomavirus Types in Multiple Infections: An Analysis in Women and Men of the High Throughput Human Papillomavirus Monitoring Study

Background

To evaluate the pattern of co-infection of human papillomavirus (HPV) types in both sexes in Sweden.

Methods

Cell samples from genital swabs, first-void urine, and genital swabs immersed in first-void urine were collected in the present cross-sectional High Throughput HPV Monitoring study. Overall, 31,717 samples from women and 9,949 from men (mean age 25) were tested for 16 HPV types using mass spectrometry. Multilevel logistic regression was used to estimate the expected number of multiple infections with specific HPV types, adjusted for age, type of sample, and accounting for correlations between HPV types due to unobserved risk factors using sample-level random effects. Bonferroni correction was used to allow for multiple comparisons (120).

Results

Observed-to-expected ratio for any multiple infections was slightly above unity in both sexes, but, for most 2-type combinations, there was no evidence of significant departure from expected numbers. HPV6/18 was found more often and HPV51/68 and 6/68 less often than expected. However, HPV68 tended to be generally underrepresented in co-infections, suggesting a sub-optimal performance of our testing method for this HPV type.

Conclusions

We found no evidence for positive or negative clustering between HPV types included in the current prophylactic vaccines and other untargeted oncogenic types, in either sex.     

From Scourge to Cure: Tumour-Selective Viral Pathogenesis as a New Strategy against Cancer

Tumour mutations corrupt cellular pathways, and accumulate to disrupt, dysregulate, and ultimately avoid mechanisms of cellular control. Yet the very changes that tumour cells undergo to secure their own growth success also render them susceptible to viral infection. Enhanced availability of surface receptors, disruption of antiviral sensing, elevated metabolic activity, disengagement of cell cycle controls, hyperactivation of mitogenic pathways, and apoptotic avoidance all render the malignant cell environment highly supportive to viral replication. The therapeutic use of oncolytic viruses (OVs) with a natural tropism for infecting and subsequently lysing tumour cells is a rapidly progressing area of cancer research. While many OVs exhibit an inherent degree of tropism for transformed cells, this can be further promoted through pharmacological interventions and/or the introduction of viral mutations that generate recombinant oncolytic viruses adapted to successfully replicate only in a malignant cellular environment. Such adaptations that augment OV tumour selectivity are already improving the therapeutic outlook for cancer, and there remains tremendous untapped potential for further innovation.     

New Insight into Neurodegeneration: the Role of Proteomics

Recent advances within the field of proteomics, including both upstream and downstream protocols, have fuelled a transition from simple protein identification to functional analysis. A battery of proteomics approaches is now being employed for the analysis of protein expression levels, the monitoring of cellular activities and for gaining an increased understanding into biochemical pathways. Combined, these approaches are changing the way we study disease by allowing accurate and targeted, large scale protein analysis, which will provide invaluable insight into disease pathogenesis. Neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), prion disease, and other diseases that affect the neuromuscular system, are a leading cause of disability in the aging population. There are no effective intervention strategies for these disorders and diagnosis is challenging as it relies primarily on clinical symptomatic features, which often overlap at early stages of disease. There is, therefore, an urgent need to develop reliable biomarkers to improve early and specific diagnosis, to track disease progression, to measure molecular responses towards treatment regimes and ultimately devise new therapeutic strategies. To accomplish this, a better understanding of disease mechanisms is needed. In this review we summarize recent advances in the field of proteomics applicable to neurodegenerative disorders, and how these advances are fueling our understanding, diagnosis, and treatment of these complex disorders.     

人乳头瘤病毒(HPV)58型DNA疫苗免疫原性的初步分析

为了检测HPV 58型不同L1基因的DNA疫苗的免疫原性,以pcDNA3.1为载体分别构建含HFV 58型不同L1基因的DNA疫苗,命名为L1h、L1h△c、L1S、L1SM和L1wt.用免疫印迹法检测各DNA疫苗的体外表达情况;各重组质粒与pcDNA3.1-h58L2和pcDNA3.1-GFP共转染293FT细胞,检测其形成假病毒的能力;并将各DNA疫苗肌肉注射免疫小鼠,利用假病毒中和实验检测中和抗体水平,用ELISPOT检测细胞免疫情况.结果显示,本实验成功构建了五种DNA疫苗,L1h△c的体外表达量最高,L1S和L1SM的表达量次之,L1wt没有表达;重组质粒L1S能够形成假病毒,而其他四种重组质粒均不能形成假病毒.L1S和L1h均可在小鼠体内诱导中和抗体,但L1S诱导的中和抗体的平均滴度为1:6 400,明显高于L1h诱导的中和抗体水平(平均滴度为1:48),而其他疫苗在小鼠体内未产生中和抗体.对五种疫苗均未检测出特异性的细胞免疫反应.结果提示,体外能够组装成假病毒的DNA疫苗在免疫动物后可诱导高滴度的中和抗体,为今后DNA疫苗的筛选提供参考.     

人乳头瘤病毒结构蛋白在病毒感染中的作用研究进展

人乳头瘤病毒(Human papillomavirus,HPV)属于乳多空病毒科(Papovaviridae)的乳头瘤病毒属,是一种无包膜的双链DNA病毒,能诱发人的皮肤或粘膜产生疣和乳头状瘤,某些基因型与子宫颈癌密切相关.     

Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.     

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis,Genome Evolution and Niche Adaptation,and Infection Detection

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V² values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates.     

Natural History of Cutaneous Human Papillomavirus (HPV) Infection in Men: The HIM Study

Accumulating evidence suggests that cutaneous human papillomavirus (HPV) infection is associated with non-melanoma skin cancer (NMSC). Little is known about the natural history of cutaneous HPV. A sub-cohort of 209 men with no NMSC history, initially enrolled in the HPV infection in men (HIM) study, were followed for a median of 12.6 months. Epidemiological data were collected through self-administered questionnaires. Cutaneous HPV DNA was measured in normal skin swabs (SS) and eyebrow hairs (EB) for 25 and 16 HPV types in genera β and γ, respectively. Any β HPV infection was more prevalent in SS (67.3%) compared to EB (56.5%, p = 0.04). Incidence in SS was higher than 20 per 1,000 person-months for HPV types 4, 5, 23, 38 and 76. Median duration of persistence of β and γ HPV infection was 8.6 and 6.1 months in EB, respectively, and 11.3 months and 6.3 months, in SS, respectively. Older age (>44 years vs. 18-30 years) was significantly associated with prevalent (SS OR = 3.0, 95% CI = 1.2–7.0) and persistent β HPV infection (EB OR = 6.1, 95% CI = 2.6–14.1). History of blistering sunburn was associated with prevalent (OR = 2.8, 95% CI = 1.3–5.8) and persistent (OR = 2.3, 95% CI = 1.2–4.6) β HPV infection in SS. Cutaneous HPV is highly prevalent in men, with age and blistering sunburn being significant risk factors for cutaneous β HPV infection.     

Vestitol as a Chemical Barrier against Intrusion of Parasitic Plant Striga hermonthica into Lotus japonicus Roots

The root parasitic plant, Striga hermonthica, constrains the production of several agronomically important poaceous crops in the arid and semiarid tropical regions of Sub-Saharan Africa. The parasite is incompatible with the model legume, Lotus japonicus. Studies at the molecular and metabolic levels have revealed that expression of the genes involved in the biosynthesis of vestitol, a legume-specific phytoalexin, was highly up-regulated in L. japonicus roots challenged with S. hermonthica. High-performance liquid chromatography and mass spectroscopy confirmed the presence of vestitol in the exudate released from L. japonicus roots inoculated with S. hermonthica seedlings. Fluorescence, similar to that emitted by authentic vestitol, was displayed on the surface of L. japonicus roots to which successful attachment of S. hermonthica had been achieved. Vestitol exerted a limited inhibitory effect on S. hermonthica germination, but it significantly inhibited seedling growth. These results indicate that vestitol biosynthesis in L. japonicus was induced by S. hermonthica attachment and that vestitol contributed, at least in part, to the host’s defence mechanism and acted as a chemical barrier against the intrusion of the parasite.     

Characterization of a New Type of Human Papillomavirus That Causes Skin Warts

A human papillomavirus (HPV) was isolated from the lesions of a patient (ML) bearing numerous hand common warts. This virus was compared with the well-characterized HPV found in typical plantar warts (plantar HPV). ML and plantar HPV DNAs have similar molecular weights (5.26 x 10(6) and 5.23 x 10(6), respectively) but were shown to be different by restriction enzyme analysis. When the cleavage products of both DNAs by endonuclease EcoRI, BamI, HpaI, or Hind were analyzed by electron microscopy, one, two, one, and four fragments were detected for ML HPV DNA instead of the two, one, two, and six fragments, respectively, detected for plantar HPV DNA. In contrast to plantar HPV DNA, a high proportion of ML HPV DNA molecules were resistant to these restriction enzymes. Most, if not all, of the molecules were either resistant to BamI and sensitive to EcoRI or sensitive to BamI and resistant to EcoRI. After denaturation and renaturation of the cleavage products of ML HPV DNA by a mixture of the two enzymes, the circular "heteroduplexes" formed showed one to three heterology loops corresponding to about 4 to 8% of the genome length. No sequence homology was detected between ML and plantar HPV DNAs by cRNA-DNA filter hybridization, by measuring the reassociation kinetics of an iodinated plantar HPV DNA in the presence of a 25-fold excess of ML HPV DNA, or by the heteroduplex technique. The two viruses had distinct electrophoretic polypeptide patterns and showed no antigenic cross-reaction by immunodiffusion or immunofluorescence techniques. Preliminary cRNA-DNA hybridization experiments, using viral DNAs from single or pooled plantar or hand warts, suggest that hand common warts are associated with viruses similar or related to ML HPV. The existence of at least two distinct types of HPVs that cause skin warts was demonstrated; they were provisionally called HPV type 1 and HPV type 2, with plantar HPV and ML HPV as prototypical viruses, respectively.     

Lability Landscape and Protease Resistance of Human Insulin Amyloid: A New Insight into Its Molecular Properties

Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8-10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.     

Functional Similarity between E6 Proteins of Cutaneous Human Papillomaviruses and the Adenovirus E1A Tumor-Restraining Module

The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins.     

Helicase-appended Topoisomerases: New Insight into the Mechanism of Directional Strand Transfer

DNA strand passage through an enzyme-mediated gate is a key step in the catalytic cycle of topoisomerases to produce topological transformations in DNA. In most of the reactions catalyzed by topoisomerases, strand passage is not directional; thus, the enzyme simply provides a transient DNA gate through which DNA transport is allowed and thereby resolves the topological entanglement. When studied in isolation, the type IA topoisomerase family appears to conform to this rule. Interestingly, type IA enzymes can carry out directional strand transport as well. We examined here the biochemical mechanism for directional strand passage of two type IA topoisomerases: reverse gyrase and a protein complex of topoisomerase IIIα and Bloom helicase. These enzymes are able to generate vectorial strand transport independent of the supercoiling energy stored in the DNA molecule. Reverse gyrase is able to anneal single strands, thereby increasing linkage number of a DNA molecule. However, topoisomerase IIIα and Bloom helicase can dissolve DNA conjoined with a double Holliday junction, thus reducing DNA linkage. We propose here that the helicase or helicase-like component plays a determinant role in the directionality of strand transport. There is thus a common biochemical ground for the directional strand passage for the type IA topoisomerases.     

Insight into the Enzyme-Inhibitor Interactions of the First Experimentally Determined Human Aromatase

Abstract

Aromatase is an important pharmacological target in the anti-cancer therapy as the intratumoral aromatase is the source of local estrogen production in breast cancer tissues. Suppression of estrogen biosynthesis by aromatase inhibition represents an effective approach for the treatment of hormone-sensitive breast cancer. Because of the membrane-bound character and heme-binding instability, no crystal structure of aromatase was reported for a long time, until recently when crystal structure of human placental aromatase cytochrome P450 in complex with androstenedione was deposited in PDB. The present study is towards understanding the structural and functional characteristics of aromatase to address unsolved mysteries about this enzyme and elucidate the exact mode of binding of aromatase inhibitors. We have performed molecular docking simulation with twelve different inhibitors (ligands), which includes four FDA approved drugs; two flavonoids; three herbal compounds and three compounds having biphenyl motif with known IC₅₀ values into the active site of the human aromatase enzyme. All ligands showed favorable interactions and most of them seemed to interact to hydrophobic amino acids Ile133, Phe134, Phe221, Trp224, Ala306, Val370, Val373, Met374 and Leu477 and hydrophilic Arg115 and neutral Thr310 residues. The elucidation of the actual structure-function relationship of aromatase and the exact binding mode described in this study will be of significant interest as its inhibitors have shown great promise in fighting breast cancer.