Hsa_circ_0001583 fuels bladder cancer metastasis by promoting staphylococcal nuclease and tudor domain containing 1-mediated MicroRNA decay.

Muscle-invasive and metastatic bladder cancer indicates extra worse prognosis. Accumulating evidence roots for the prominent role of circular RNAs(circRNAs) in bladder cancer, while the mechanisms linking circRNAs and bladder cancer metastasis remain limitedly investigated. Here, we identified a significantly upregulated circRNA candidate, hsa_circ_0001583, from online datasets. Validated by qRT-PCR, PCR, sanger sequencing, actinomycin D and RNase R digestion experiments, hsa_circ_0001583 was proved to be a genuine circular RNA with higher expression levels in bladder cancer tissue. Through gain and loss of function experiments, hsa_circ_0001583 exhibited potent migration and invasion powers both in vitro and in vivo. The staphylococcal nuclease and Tudor domain containing 1 (SND1) was identified as an authentic binding partner for hsa_circ_0001583 through RNA pulldown and RIP experiments. Elevated levels of hsa_circ_0001583 could bind more to SND1 and protect the latter from degradation. Rescue experiments demonstrated that such interaction-induced increased in SND1 levels in bladder cancer cells enabled the protein to pump its endonuclease activity, leading to the degradation of tumor-suppressing MicroRNAs (miRNAs) including miR-126-3p, the suppressor of Disintegrin And Metalloproteinase Domain-Containing Protein 9 (ADAM9), ultimately driving cells into a highly migrative and invasive state. In summary, our study is the first to highlight the upregulation of hsa_circ_0001583 in bladder cancer and its role in downregulating miR-126-3p by binding to and stabilizing the SND1 protein, thereby promoting bladder cancer cell migration and invasion. This study adds hsa_circ_0001583 to the pool of bladder cancer metastasis biomarkers and therapeutic targets.

Therapeutic effects of duck Tembusu virus capsid protein fused with staphylococcal nuclease protein to target Tembusu infection in vitro.

Tembusu virus (TMUV), a member of the genus flavivirus, primarily causes egg-drop syndrome in ducks and is associated with low disease mortality but high morbidity. The commercially available live vaccines for treating TMUV currently include the main WF100, HB, and FX2010-180P strains, and efficient treatment and/or preventative measures are still urgently needed. Capsid-targeted viral inactivation (CTVI) is a conceptually powerful new antiviral strategy that is based on two proteins from the capsid protein of a virus and a crucial effector molecule. The effector molecule can destroy the viral DNA/RNA or interfere with the proper folding of key viral proteins, while the capsid protein mainly plays a role in viral integration and assembly; the fusion proteins are incorporated into virions during packaging. This study aimed to explore the potential use of this strategy in duck TMUV. Our results revealed that these fusion proteins can be expressed in susceptible BHK21 cells without cytotoxicity and possess excellent Ca2+-dependent nuclease activity, and their expression is also detectable in DF-1 cells. Compared to those in the negative controls (BHK21 and BHK21/pcDNA3.1(+) cells), the numbers of viral RNA copies in TMUV-infected BHK21/Cap-SNase and BHK21/Cap-Linker-SNase cells were reduced by 48 h, and the effect of Cap-Linker-SNase was superior to that of Cap-SNase. As anticipated, these results suggest that these fusion proteins contribute to viral resistance to treatment. Thus, CTVI might be applicable for TMUV inhibition as a novel antiviral therapeutic candidate during viral infection.

Oral delivery of staphylococcal nuclease by Lactococcus lactis prevents type 1 diabetes mellitus in NOD mice.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the self-destruction of insulin-producing ß cells. Recently, studies have revealed that neutrophils contribute to the early pathological injury to the pancreas, predominantly via the formation of neutrophil extracellular traps (NETs). To determine whether early intervention targeting NETs with staphylococcal nuclease (SNase) can delay the onset of T1DM, non-obese diabetic (NOD) mice were orally administered recombinant Lactococcus lactis (L. lactis) expressing SNase. The results showed that NETs were effectively disrupted by SNase both in vivo and in vitro, leading to a significant decrease in neutrophil-derived circulating free DNA (cf-DNA/NETs), neutrophil elastase (NE), and protease 3 (PR3) in the serum compared with the controls. In addition, SNase effectively regulated the blood glucose levels of NOD mice, and the onset of diabetes was postponed with reduced mortality and morbidity. Recombinant L. lactis also ameliorated inflammation in NOD mice, as evidenced by the remarkable increase in IL-4 and reductions in TNF-α and CRP. Moreover, HE staining results showed that L. lactis expressing SNase exerted protective effects on pancreatic islets and relieved inflammation of the small intestine in NOD mice. Hence, the present study indicates that the oral delivery of SNase by L. lactis can effectively prevent T1DM, ameliorate inflammation, and contribute to immunomodulatory balance in NOD mice.

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid.

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.

Inhibition of DNA replication of human papillomavirus by using zinc finger-single-chain FokI dimer hybrid.

Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.

Nonuniform chain collapse during early stages of staphylococcal nuclease folding detected by fluorescence resonance energy transfer and ultrarapid mixing methods.

The development of tertiary structure during folding of staphylococcal nuclease (SNase) was studied by time-resolved fluorescence resonance energy transfer measured using continuous- and stopped-flow techniques. Variants of this two-domain protein containing intradomain and interdomain fluorescence donor/acceptor pairs (Trp and Cys-linked fluorophore or quencher) were prepared to probe the intradomain and interdomain structural evolution accompanying SNase folding. The intra-domain donor/acceptor pairs are within the ß-barrel domain (Trp27/Cys64 and Trp27/Cys97) and the interdomain pair is between the α-helical domain and the ß-barrel domain (Trp140/Cys64). Time-resolved energy transfer efficiency accompanying folding and unfolding at different urea concentrations was measured over a time range from 30 µs to ≈ 10 s. Information on average donor/acceptor distances at different stages of the folding process was obtained by using a quantitative kinetic modeling approach. The average distance for the donor/acceptor pairs in the ß-barrel domain decreases to nearly native values whereas that of the interdomain donor/acceptor pairs remains unchanged in the earliest intermediate (<500 µs of refolding). This indicates a rapid nonuniform collapse resulting in an ensemble of heterogeneous conformations in which the central region of the ß-barrel domain is well developed while the C-terminal α-helical domain remains disordered. The distance between Trp140 and Cys64 decreases to native values on the 100-ms time scale, indicating that the α-helical domain docks onto the preformed ß-barrel at a late stage of the folding. In addition, the unfolded state is found to be more compact under native conditions, suggesting that changes in solvent conditions may induce a nonspecific hydrophobic collapse.

Molecular characterization of Staphylococcus aureus from patients with surgical site infections at Mulago Hospital in Kampala, Uganda.

BACKGROUND: The prevalence of Methicillin resistant Staphylococcus aureus (MRSA) is progressively increasing globally with significant regional variation. Understanding the Staphylococcus aureus lineages is crucial in controlling nosocomial infections. Recent studies on S. aureus in Uganda have revealed an escalating burden of MRSA. However, the S. aureus genotypes circulating among patients are not known. Here, we report S. aureus lineages circulating in patients with surgical site infections (SSI) at Mulago National hospital, Kampala, Uganda. METHODS: A cross-sectional study involving 314 patients with SSI at Mulago National Hospital was conducted from September 2011 to April 2012. Pus swabs from the patients' SSI were processed using standard microbiological procedures. Methicillin sensitive Staphylococcus aureus (MSSA) and MRSA were identified using phenotypic tests and confirmed by PCR-detection of the nuc and mecA genes, respectively. SCCmec genotypes were determined among MRSA isolates using multiplex PCR. Furthermore, to determine lineages, spa sequence based-genotyping was performed on all S. aureus isolates. RESULTS: Of the 314 patients with SSI, S. aureus accounted for 20.4% (64/314), of which 37.5% (24/64) were MRSA. The predominant SCCmec types were type V (33.3%, 8/24) and type I (16.7%, 4/24). The predominant spa lineages were t645 (17.2%, 11/64) and t4353 (15.6%, 10/64), and these were found to be clonally circulating in all the surgical wards. On the other hand, lineages t064, t355, and t4609 were confined to the obstetrics and gynecology wards. A new spa type (t10277) was identified from MSSA isolate. On multivariate logistic regression analysis, cancer and inducible clindamycin resistance remained as independent predictors of MRSA-SSI. CONCLUSION: SCCmec types I and V are the most prevalent MRSA mecA types from the patients' SSI. The predominant spa lineages (t645 and t4353) are clonally circulating in all the surgical wards, calling for strengthening of infection control practices at Mulago National Hospital.

Gene- and protein-delivered zinc finger-staphylococcal nuclease hybrid for inhibition of DNA replication of human papillomavirus.

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

Mutational analysis of m-values as a strategy to identify cold-resistant substructures of the protein ensemble.

Characterizing the native ensemble of protein is an important yet difficult objective of structural biology. The structural dynamics of protein macromolecules play key roles in biological function, but the short lifetimes and low population of near-native states of the protein ensemble limit their ability to be studied directly. In part to address such issues, it was shown recently that the cooperative substructures that populate a protein ensemble could be ascertained by NMR methods performed at very cold temperatures. What is presented here is an argument that these same substructures can also be determined by denaturant-induced unfolding studies performed on protein at room temperature. Data supporting this argument are given for Staphylococcal nuclease, chymotrypsin inhibitor 2, and ubiquitin. The observation of an agreement between the thermodynamics of the protein ensemble simulated under very cold temperatures to the apparent sensitivity of the ensemble to chemical denaturants at room temperature also suggests that the overall structural-thermodynamic character of an ensemble is surprisingly robust and preserved even in the presence of strong denaturing conditions.

Nasal carriage of Panton Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus in healthy preschool children / Colonización nasal de Staphylococcus aureus Meticilino Resistente portador de la Leucocidina Panton-Valentine en preescolares

Objective Determining the prevalence of nasal carriage of S. aureus, both sensitive to methicillin and resistant to it, in preschool children and evaluating the presence of Panton-Valentine leukocidin genes in the isolates. Methods This was a cross-sectional study in which cultures from anterior nares were obtained from healthy preschool children. Isolates were identified as S. aureus based on morphological and biochemical tests. Antibiotic susceptibility profiles were determined by the disk diffusion method. All the isolates were further analyzed by multiplex PCR to determine the presence of mecA and PVL genes; methicillin-resistant isolates were also SCCmec typed by multiplex PCR. Results Overall S. aureus nasal colonization prevalence was 38.5 % and 4.8 % for methicillin-resistant strains. All the methicillin-resistant isolates carried the genes for PVL; two isolates possessed the SCCmec type IV, two were SCCmec type I and one was SCCmec type II. Conclusion This study revealed high PVL-positive, methicillin-resistant S. aureus colonization prevalence in healthy preschool children from Cartagena, which may play a key role in the epidemiology of community-associated infection by methicillin-resistant S. aureus in healthy children from this particular geographical area.

Objetivo Determinar la prevalencia de colonización nasal de S. aureus, tanto sensible como resistente a meticilina, en niños preescolares y evaluar la presencia de los genes de la leucocidina Panton-Valentine en estos aislamientos. Métodos Estudio de corte transversal en el que se realizaron cultivos de flora nasal de niños preescolares. Los aislamientos fueron identificados como S. aureus con base en su morfología y pruebas bioquímicas. La susceptibilidad a antibióticos se determinó por el método de difusión en disco. Todos los aislamientos fueron analizados por PCR múltiple para determinar la presencia de los genes mecA y PVL, y para la tipificación del casete cromosómico SCCmec de los aislamientos resistentes a meticilina. Resultados La colonización nasal por S. aureus fue 38,5 %, y la de cepas meticilino-resistentes fue 4,8 %. Todos los aislamientos SARM portaban los genes para PVL, dos portaban el elemento SCCmec tipo IV, dos fueron tipo I y uno fue tipo II. Conclusión Encontramos una alta prevalencia de colonización por cepas meticilino-resistentes, PVL-positivos en la población estudiada, lo que podría jugar un papel clave en la epidemiología de las infecciones por S.aureus meticilino-resistente en esta área geográfica.

Nonlocal interactions are responsible for tertiary structure formation in staphylococcal nuclease.

Rapid molecular collapse mediated by nonlocal interactions is believed to be a crucial event for protein folding. To investigate the role of nonlocal interactions in tertiary structure formation, we performed a nonlocal interaction substitution mutation analysis on staphylococcal nuclease (SNase). Y54 and I139 of wild-type (WT) SNase and Delta140-149 were substituted by cysteine to form intramolecular disulfide bonds, respectively called WT-SS and Delta140-149-SS. Under physiological conditions, the reduced form of Delta140-149-SS appears to assume a denatured structure; in contrast, the oxidized form of Delta140-149-SS forms a native-like structure. From this result, we conclude that the C-terminal region participates in a nonlocal interaction that is indispensable for the native structure. Although the oxidized form of WT-SS assumes a more compact denatured structure under acidic conditions than the WT, the kinetic measurements reveal that the refolding reactions of both the reduced and oxidized forms of WT-SS are similar to those of the WT, suggesting that an intact nonlocal interaction is established within the dead time (22 ms). On the basis of these results, we propose that the native nonlocal contact established at the early stage of the folding process facilitates further secondary structure formation.

Effects of excluded volume upon protein stability in covalently cross-linked proteins with variable linker lengths.

To explore the effects of molecular crowding and excluded volume upon protein stability, we used a series of cross-linking reagents with nine different single-cysteine mutants of staphylococcal nuclease to make covalently linked dimers. These cross-linkers ranged in length from 10.5 to 21.3 A, compelling separations which would normally be found only in the most concentrated protein solutions. The stabilities of the dimeric proteins and monomeric controls were determined by guanidine hydrochloride and thermal denaturation. Dimers with short linkers tend to exhibit pronounced three-state denaturation behavior, as opposed to the two-state behavior of the monomeric controls. Increasing linker length leads to less pronounced three-state behavior. The three-state behavior is interpreted in a three-state model where cross-linked native protein dimer, N-N, interconverts in a two-state transition with a dimer where one protein subunit is denatured, N-D. The remaining native protein in turn can denature in another two-state transition to a state, D-D, in which both tethered proteins are denatured. Three-state behavior is best explained by excluded volume effects in the denatured state. For many dimers, linkers longer than 17 A removed most three-state character. This sets a limit on the flexibility and size of the denatured state. Notably, in contradiction to theoretical predictions, these cross-linked dimers were not stabilized. The failure of these predictions is possibly due to neglect of the alteration in hydrophobic exposure that accompanies any significant reduction in the conformational space of the denatured state.

Tudor domain proteins in protozoan parasites and characterization of Plasmodium falciparum tudor staphylococcal nuclease.

RNA-binding proteins play key roles in post-transcriptional regulation of gene expression. In eukaryotic cells, a multitude of RNA-binding proteins with several RNA-binding domains/motifs have been described. Here, we show the existence of two Tudor domain containing proteins, a survival of motor neuron (SMN)-like protein and a Staphylococcus aureus nuclease homologue referred to as TSN, in Plasmodium and other protozoan parasites. Activity analysis shows that Plasmodium falciparum TSN (PfTSN) possesses nuclease activity and Tudor domain is the RNA-binding domain. A specific inhibitor of micrococcal nucleases, 3',5'-deoxythymidine bisphosphate (pdTp) inhibits the nuclease as well as RNA-binding activities of the protein. PfTSN shows a predominant nuclear localization. Treatment of P. falciparum with pdTp, inhibited in vitro growth of both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, while a four fold concentration of pdTp did not have any significant effect on the mammalian cell line, Huh-7D12. Altogether, these results suggest that PfTSN is an essential enzyme in the parasite's life cycle.

[Investigation of the presence of panton-valentin leucocidin (PVL) in Staphylococcus aureus strains isolated from clinical samples]. / Klinik örneklerden izole edilen Staphylococcus aureus suslarinda panton-valentin lökosidin (PVL) varliginin arastirilmasi.

Panton-Valentin leucocidin (PVL) is a cytotoxin which causes tissue necrosis by degradating leucocytes and other cell types. PVL has recently become very up to date as it has been shown to be the major virulance factor of community acquired methicillin resistant Staphylococcus aureus strains. In this study, the presence of PVL was investigated in methicillin sensitive and resistant S. aureus (MSSA and MRSA, respectively) strains which were isolated from clinical samples between January 2005-May 2006 at Dokuz Eylul University Hospital, Izmir. Fifty five MRSA and 79 MSSA strains which were isolated from blood, wound and respiratory tract samples were randomly included to the study. The presence of PVL was evaluated by multiplex polymerase chain reaction (PCR) which detects pvl and S. aureus-specific nuc genes. As a result, PVL positivities were detected in two (5%) of 40 MSSA and four (10.3%) of 39 MSSA strains isolated in the years 2005 and 2006, respectively. None of the MRSA isolates had pvl gene. Although this cytotoxin was rarely detected among MSSA isolates, it was interesting to note that the prevalence of PVL was twice more in the year 2006 compared to 2005. It was also worth to notify that four of six (66.7%) PVL positive strains had been isolated from the patients of general surgery inpatient or outpatient clinics.

The RISC subunit Tudor-SN binds to hyper-edited double-stranded RNA and promotes its cleavage.

Long perfect double-stranded RNA (dsRNA) molecules play a role in various cellular pathways. dsRNA may undergo extensive covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), resulting in conversion of up to 50% of adenosine residues to inosine (I). Alternatively, dsRNA may trigger RNA interference (RNAi), resulting in silencing of the cognate mRNA. These two pathways have previously been shown to be antagonistic. We show a novel interaction between components of the ADAR and RNAi pathways. Tudor staphylococcal nuclease (Tudor-SN) is a subunit of the RNA-induced silencing complex, which is central to the mechanism of RNAi. Here we show that Tudor-SN specifically interacts with and promotes cleavage of model hyper-edited dsRNA substrates containing multiple I.U and U.I pairs. This interaction suggests a novel unsuspected interplay between the two pathways that is more complex than mutual antagonism.

Sensitivity of NMR residual dipolar couplings to perturbations in folded and denatured staphylococcal nuclease.

The invariance of NMR residual dipolar couplings (RDCs) in denatured forms of staphylococcal nuclease to changes in denaturant concentration or amino acid sequence has previously been attributed to the robustness of long-range structure in the denatured state. Here we compare RDCs of the wild-type nuclease with those of a fragment that retains a folded OB-fold subdomain structure despite missing the last 47 of 149 residues. The RDCs of the intact protein and of the truncation fragment are substantially different under conditions that favor folded structure. By contrast, there is a strong correlation between the RDCs of the full-length protein and the fragment under denaturing conditions (6 M urea). The RDCs of the folded and unfolded forms of the proteins are uncorrelated. Our results suggest that RDCs are more sensitive to structural changes in folded than unfolded proteins. We propose that the greater susceptibility of RDCs in folded states is a consequence of the close packing of the polypeptide chain under native conditions. By contrast, the invariance of RDCs in denatured states is more consistent with a disruption of cooperative structure than with the retention of a unique long-range folding topology.

Thermonuclease gene as a target for specific identification of Staphylococcus intermedius isolates: use of a PCR-DNA enzyme immunoassay.

A PCR-DNA enzyme immunoassay (PCR-DEIA) was developed for identification of the coagulase-positive species Staphylococcus intermedius. Two PCR primers and a hybridization probe were designed to target specific sequences of the S. intermedius thermonuclease (nuc) gene. In addition to S. intermedius reference strains, the PCR-DEIA was tested using 295 veterinary and human S. intermedius isolates. A specific 933-bp DNA fragment was successfully amplified in 281 (94.9%) S. intermedius isolates. Five canine isolates showed an unexpected 2.8-kbp band. Except for 10 amplicons derived from equine, camel, and pigeon isolates, all positive PCR results (n = 288, 96.6%) were confirmed by the colorimetric microtiter plate DEIA hybridization. Isolates that failed both in amplification and DEIA hybridization were only observed in equine isolates (10/23, 43.5%). Except for the limitations with isolates of hoofed animals, the S. intermediusnuc PCR assay has potential for rapid identification of S. intermedius and differentiation from other coagulase-positive staphylococci including S. aureus.

The transmembrane domains of ErbB receptors do not dimerize strongly in micelles.

The epidermal growth factor receptors (erbB) constitute an important class of single pass transmembrane receptors involved in the transduction of signals important for cell proliferation and differentiation. Receptor association is a key event in the signal transduction process, but the molecular basis of this interaction is not fully understood. Previous biochemical and genetic studies have suggested that the single transmembrane helices of these receptor proteins might play a role in stabilizing the receptor complexes. To determine if the erbB transmembrane domains could provide a driving force to stabilize the receptor dimers, we carried out a thermodynamic study of these domains expressed as C-terminal fusion proteins with staphylococcal nuclease. Similar fusion constructs have been used successfully to investigate the oligomerization and association thermodynamics of a number of transmembrane sequences, including that of glycophorin A. Using SDS-PAGE analysis and sedimentation equilibrium analytical ultracentrifugation, we do not find strong, specific homo or hetero-interactions between the transmembrane domains of the erbB receptors in micellar solutions. Our results indicate that any preferential interactions between these domains in micellar solutions are extremely modest, of the order of 1 kcal mol(-1) or less. We applied a thermodynamic formalism to assess the effect of weakly interacting TM segments on the behavior of a covalently attached soluble domain. In the case of the ligand-bound EGFR ectodomain, we find that restriction of the ectodomain to the micellar phase by a hydrophobic TM, even in the absence of strong specific interactions, is largely sufficient to account for the previously reported increase in dimerization affinity.

Neural network-based prediction of mutation-induced protein stability changes in Staphylococcal nuclease at 20 residue positions.

Protein-based therapeutics are playing an increasingly important role in the treatment of diseases, including diabetes and cancer. The viability of these treatments, however, are highly dependent on the stability of the therapeutic, since stability affects both the shelf life of the therapeutic as well as its active life in the body. Stability engineering can, therefore, be used to increase the effectiveness of protein-based therapeutics. Computational methods of protein stability prediction have been under development for about a decade, but complex molecular interactions make stability prediction difficult and computationally intensive. A rapid computational method of protein stability prediction is developed using feed-forward neural networks and used to predict mutation-induced stability changes in Staphylococcal nuclease. The input to the neural network consisted of sequences of evolutionarily based amino acid similarity scores that were obtained through the comparison of the amino acids in a mutation containing sequence to their positional counterparts in the baseline wild-type amino acid sequence. A training set was created which consisted of similarity score sequences, for which the stabilities of the corresponding amino acid sequences were known, paired with the relative stabilities of the sequences to that of the baseline. Back-propagation of error was used to train the network to output accurate relative stability scores for the sequences in the training set. Neural network-based relative stability predictions for 55 sequences containing mutation combinations not found in the training set had an accuracy of 92.8%.

Therapeutic effects of dengue 2 virus capsid protein and staphylococcal nuclease fusion protein on dengue-infected cell cultures.

Dengue infection poses a serious public health problem in most tropical and subtropical areas. No effective antiviral drugs or vaccines are currently available against dengue infection. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection, we constructed a plasmid expressing a fusion protein consisting of staphylococcal nuclease (SN) fused to dengue 2 virus capsid protein (D2C), and investigated its effects on the production of infectious virions when introduced into BHK cells infected with dengue virus. The results indicated that D2C-SN can be expressed and tolerated in this mammalian cell culture. The enzymatically active SN moiety was incorporated into nascent virions during the process of viral assembly. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of wild-type SN, on the infectivity of progeny virions, we clearly demonstrated that nucleolytic activity was the major antiviral mechanism. Expression of D2C-SN fusion protein as a therapeutic agent resulted in a reduction in infectious titers of 12- to 60-fold. Therefore, dengue virus may be particularly vulnerable to a CTVI therapeutic approach.