Month: September 2020

T4 head-decorating proteins Hoc and Soc do not cause any changes in the Major Capsid Protein arrangement, and their presence is not essential for phage viability. These two proteins have been successfully applied in phage display of foreign proteins and peptides on T4 capsid, mostly as novel vaccine platforms but also for phage purification. Gphoc and gpsoc have also been shown to influence T4 phage susceptibility to cellular uptake. We developed procedures that allow us to obtain highly purified native phage proteins suitable for immunological assays both in vivo and in vitro. Furthermore, we analysed the secondary structure of these proteins by circular dichroism spectroscopy. We also determined the proteins’ stability in increasing concentrations of guanidine chloride. In this work we optimized expression and purification of four bacteriophage T4 head proteins in E. coli, and obtained their immunological purity grade preparations. As was known before, the major head protein gp23 requires assistance of the phage-encoded chaperonin gp31 that is a functional analogue of bacterial GroES protein able to functionally replace it in interaction with the cellular chaperonin GroEL. However, in our experiments this protein was also GW-572016 produced in native conformation using low temperature expression in presence of Cpn10 and Cpn60 chaperones derived from Oleispira antarctica showing 74 and 54% amino acid identity with groEL and groES of E. coli. Recently, the ability of groEL-groES complex to interact with gp23 was reported. Low temperature expression without chaperones yielded an insoluble product, which leads to the suggestion that Cpn10–Cpn60 complex may possess some feature of GroEL-gp31 complex or groEL–groES complex allowing it to assist gp23 folding in T4 infected cells. However, from the practical point of view the best way to produce native gp23 is its co-expression with gp31 protein naturally assisting its folding. Unexpectedly, effective production of gpsoc, the smallest protein in the T4 head, was difficult due to its poor yield when N-terminal fusion of the affinity tag was used. It is possible that some specific sequences present at the gpsoc C-terminus directs this protein to degradation by bacterial proteases. Therefore the soc gene was re-cloned into the expression vector that allowed its expression with a C-terminal affinity tag, resulting in good yield of the protein, but it was almost insoluble. Enhanced expression of chaperone TF improved the solubility to a sufficient level. This result raises a question on the folding of this protein in natural T4 infection. The optimised purification procedure allows for production of nearly homogeneous preparations of all four proteins with very low LPS activity. These proteins have proven native structure and are sufficiently stable, which makes them applicable for immunological studies in vitro and in vivo. Non-essential decorating gphoc represents a typical beta-type secondary structure. This structure has been investigated by in a T4-like bacteriophage, RB49, whose gphoc has only 22% sequence identity with T4.

Laevis will lead to a better understanding of the biological functions, expression regulation mechanisms, pathogenicity and other important aspects of GOLPH2. The protein universe refers to a collection of all proteins across all organisms in nature. In 1992, there were only 887 protein structures in the Protein Data Bank which could be categorized into 120 different tertiary folds. Chothia noticed that about 1/4 of the entries at the EMBL/SwissProt sequence databank were homologous to the 120 folds, and 1/3 of the genome sequences presented in the sequence databank. He thereby suggested that the number of protein tertiary folds in the protein universe XL880 cost should be limited and around 1500. Amazingly, this simple estimation stood well the test of time and lies at the center of the subsequent estimation range using more elaborate methods based on much larger datasets.

At present, the PDB has over 70 k structures, which has been argued to be structurally complete. The structure set has been categorized into 1,195 folds by SCOP in the 2009 release, consistent with the Chothia’s original estimation. In contrast to the extensive studies of protein tertiary structural space, the quaternary structure space of protein-protein interactions is relatively unexplored. For example, the questions on whether the number of unique protein-protein complex structures is constrained and if yes, how many they are, have remained largely unanswered. Since most proteins perform their physiological functions via interaction with other protein molecules, the answers to these questions have practical applications in the understanding of protein-protein interaction specificity and protein-protein networks. Meanwhile, the template-based methods have recently demonstrated promising power in protein complex structural modeling; the completeness of the quaternary structure space is of important implications to the studies of protein-protein docking and structure prediction, and the forthcoming structural genomics of protein-protein interactions. Exploration of the quaternary structure space has been mainly hampered by the relative dearth of protein-protein complex structures in the PDB library, and the lack of an unambiguous definition of protein quaternary structural folds and efficient methods to compare and categorize protein-protein complex structures. Among limited attempts, Aloy and Russell exploited the protein-protein interaction data from high-throughput genomic data to estimate, based on the assumption that homologous proteins should participate in similar interactions, that the number of unique protein-protein interactions is around 10,000.

Although the estimation could be meaningful for the complex homologous families, it is often observed that proteins of different sequences have similar complex structure and interface interactions. Thus, the AloyRussell calculation may overestimate the protein-protein interaction space if the protein-protein interactions are counted at the structural level. Here, we present a systemic study of a representative set of protein-protein complex structures in the PDB.

Small GTP-binding proteins are monomeric G proteins with molecular masses of 20–40 kDa. Small G proteins in eukaryotes from yeast to human constitute a superfamily with at least five families including more than 100 members. Although plants have only four of these families of small G proteins, they have a unique subfamily of Rho GTPases instead of the Ras family. Rac1, a member of the Rho family, has been shown to play an essential role in the defense of rice against pathogens. The functions of other superfamilies of small G proteins in plant toward pathogens have not been determined. The Rab proteins belong to the small guanosine triphosphatases superfamily. Rabs are thought to act as molecular switches, which play an essential role in both endocytic and exocytic traffic in eukaryotic cells, being active in their GTPbound state and inactive in their GDC-0879 GDP-bound state.

Because Rabs do not have high intrinsic guanine nucleotide exchange or hydrolytic activities, they are regulated by other proteins, such as guanine nucleotide exchange factors and GTPase-activating proteins. In their GDP-bound state, Rabs are typically soluble and bound to guanine nucleotide dissociation inhibitor. At the acceptor membrane, the Rab-GDI complex is thought to interact with GDI displacement factor, which removes GDI and allows Rab membrane insertion. Next, a GEF converts Rab to its GTP-bound, active conformation, allowing it to interact with its downstream effectors. Rabs regulate cell proliferation, cytoskeleton organization, intracellular membrane trafficking and vesicle motility along the actin/microtubule cytoskeletons, vesicle tethering, transport, and fusion. Many downstream effectors of Rab7 in mammals have been extensively characterized and shown to interact with their partners to exert biological functions. Rab7 is a key regulator in the process of phagosome maturation. Rab7 and the Rab interacting lysosomal protein are essential factors in regulating the maturation of the phagosome into a lysophagosome. In addition, the homotypic fusion and vacuole protein sorting may play dual roles as upstream GEF and downstream tethering effector of Rab7 to facilitate endosomal membrane fusion.

Approximately 70 members have been identified in mammals, and Rab7 is one of the Rab proteins that have been investigated extensively. Rab7 is regarded as a key regulator in endo-lysosomal trafficking based on the extensive investigations in the past decades. Rab7 mediates the regulated internalization and degradation of nutrient transporters and triggers nutrient starvation that facilitates cell death. A tonoplast-localized rice Rab7 homologue is up-regulated in response to cold, salt, and drought stress, suggesting that it plays a role in plant adaptation to various environmental stresses. In Arabidopsis, Kwon et al. analyzed a SA-responsive protein involved in plant-pathogen interactions RabG3b, which shows strong similarity to the Rab7 in mammals and yeast which contain conserved motifs for GTP binding and hydrolysis. Compared to wild-type and transgenic plants overexpressing dominant negative RabG3b, transgenic plants overexpressing.

As EGFR was found to induce miR-7 expression through a Ras/ ERK/Myc pathway, which promoted cell growth and tumor formation, indicating an indirect feedback loop. The expression of the EGFR has also been negatively associated with the expression of miR-30c, which was, identified as an independent predictor of outcome in advanced breast cancer; high expression of miR-30c was associated with benefit of Tamoxifen. Cross-talk between ER and EGFR and its family of growth factor receptors, especially in its activated phosphorylated form, has also been shown to be strongly associated with Tamoxifen resistance.

We previously conducted a study of mRNA expression in patient samples from the discovery set and found no significant association between EGFR mRNA levels and tumor grade, but a significant association with outcome. Furthermore, using the same patient material, a significant correlation between the level of pEGFR and outcome was also observed on a tissue microarray, but again, no correlation with grade was observed. It is well-known that tumor grade is a predictor of poor outcome. The cause of this lack of consistency between the miRNA and mRNA studies with regards to associations to outcome may reflect the complexity of the biological system with multiple miRNAs per target, and that miRNAs have several targets. Furthermore, the apparent indirect feedback loop between miR-7 and EGFR is affected by several other factors, likely masking the known association with grade to outcome. The miRNAs that exhibited the most variable expression in the individual set and overlapped across the three sets were selected, yielded a set of 31 miRNAs. Since the discussed miRNAs were identified by unsupervised analysis, whether they merely depict the cellular architecture of the tumors or are actually masked markers for the tumors ability to progress despite adjuvant treatment with a target-specific drug remains to be determined.

One fourth of the 31 miRNAs were found to be associated with EMT/MET and stem cell characteristics, and the notion that cancer stem cells play an important role in endocrine resistance has recently been discussed. Further studies into this field are needed. It would also be interesting to conduct in situ hybridization of the identified miRNAs to visualize the cells they are expressed in. Also, functional studies would clarify whether targeting surrounding tissue would induce tumor regression, since it is well-known that the microenvironment and malignant cells interact. Overall, the number of patients in this study provides a strong foundation for the debate on miRNAs and benefit of Tamoxifen in post-menopausal ER+ breast cancer patients. This global analysis, employing the microarray-technique, allows a general estimation of miRNAs potential and is a common XL-184 849217-68-1 hypothesis-generating method. This also implies, however, that the significance of single miRNAs expressed by a subgroup of patients with yet unknown characteristics may be masked when assessing the groups collectedly as only general differences are detected. In conclusion, our data seems to indicate that no single miRNA profile predictive of outcome for tumors from ER+ breast cancer patients receiving adjuvant Tamoxifen mono-therapy can be identified.

MiRNAs have been suggested as promising biomarkers, including potential markers of Tamoxifen resistance. Herein we report the first large-scale global miRNA expression study on primary tumors from high-risk, ER+, post-menopausal breast cancer patients who received adjuvant Tamoxifen mono-therapy. Our study showed that although a highly significant set of 10 miRNAs distinguishing patient samples according to outcome were identified in the discovery set, the miRNA profile could not be confirmed in the subsequent two independent test sets. Our data indicate that there is likely no single, strongly predictive profile of outcome, and even small imbalances with regard to prognostic factors between the recurrent/non-recurrent groups, such as number of tumor-infiltrated lymph nodes at time of diagnosis, may mask identification of potentially predictive miRNAs. Further, it may be speculated that endocrine resistance occurs as a result of several different mechanism, each employing a set of unique miRNA, complicating the identification of a common set of miRNAs that is altered in primary tumors of breast cancer patients who do not have the benefit of adjuvant Tamoxifen treatment. This finding is somewhat surprising, since several miRNAs have been suggested to be associated with Tamoxifen resistance. However, the majorities of these studies were performed in cell line models or used very limited numbers of patient samples. Moreover, several studies identified miRNAs involved in ER regulation, and thus only indirectly proposed to be involved in Tamoxifen resistance. One of these, the miR-221/-222 cluster, has been found to be in a feedback loop with ERa that results in a decrease in ERa protein, which can lead to resistance towards Tamoxifen. The reason for not identifying this miRNA cluster in our study may be related to the fact that most of the patient tumor samples we analyzed exhibited a very high percentage of ERa positive cells, with a median of 83%, across the three sets. Another miRNA, miR-210, was identified in a dataset of untreated patients consisting of 25 ER-, 40 ER+ and 8 ERunknown tumor samples, and therefore likely represents a general marker for outcome, whereas our study focused on resistance towards Tamoxifen from the onset. Another recent large study could also not confirm the association of miR-210 with outcome. The discovery set was clinically very homogenous, and the Nand R-groups were nearly identical according to the strong prognostic factors that influence the likelihood of developing recurrence. The two test sets, on the other hand, had more variable clinical characteristics per group. Where the R-group had an average of 8.6, whereas the N-group had 3.0. For test set #2, there was a minor difference in the average tumor size and in tumorinfiltrated lymph nodes, but there was also variation in the duration of treatment with Tamoxifen. The two test sets were randomly selected, as opposed to the discovery set, which was matched, and potential biomarkers, such as miRNAs, would need the capacity to overcome patient-to-patient variation to achieve clinical value. Thus, the miRNA profile does not seem to provide information with regard to the probability of GSK2118436 1195765-45-7 recurrence following adjuvant Tamoxifen-treatment in post-menopausal ER breast cancer patients.