Expression, purification, and characterisation of the p53 binding domain of Retinoblastoma binding protein 6 (RBBP6).

RBBP6 is a 250 kDa eukaryotic protein known to be a negative regulator of p53 and essential for embryonic development. Furthermore, RBBP6 is a critical element in carcinogenesis and has been identified as a potential biomarker for certain cancers. RBBP6's ability to interact with p53 and cause its degradation makes it a potential drug target in cancer therapy. Therefore, a better understating of the p53 binding domain of RBBP6 is needed. This study presents a three-part purification protocol for the polyhistidine-tagged p53 binding domain of RBBP6, expressed in Escherichia coli bacterial cells. The purified recombinant domain was shown to have structure and is functional as it could bind endogenous p53. We characterized it using clear native PAGE and far-UV CD and found that it exists in a single form, most likely monomer. We predict that its secondary structure is predominantly random coil with 19% alpha-helices, 9% beta-strand and 14% turns. When we exposed the recombinant domain to increasing temperature or known denaturants, our investigation suggested that the domain undergoes relatively small structural changes, especially with increased temperature. Moreover, we notice a high percentage recovery after returning the domain close to starting conditions. The outcome of this study is a pure, stable, and functional recombinant RBBP6-p53BD that is primarily intrinsically disordered.

Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies.

Cancer is a complex disease whereby multiple genetic aberrations, epigenetic modifications, metabolic reprogramming, and the microenvironment contribute to the development of a tumor. In the traditional anticancer drug discovery pipeline, drug candidates are usually screened in vitro using two-dimensional or three-dimensional cell culture. However, these methods fail to accurately mimic the human disease state. This has led to the poor success rate of anticancer drugs in the preclinical stages since many drugs are abandoned due to inefficacy or toxicity when transitioned to whole-organism models. The common fruit fly, Drosophila melanogaster, has emerged as a beneficial system for modeling human cancers. Decades of fundamental research have shown the evolutionary conservation of key genes and signaling pathways between flies and humans. Moreover, Drosophila has a lower genetic redundancy in comparison to mammals. These factors, in addition to the advancement of genetic toolkits for manipulating gene expression, allow for the generation of complex Drosophila genotypes and phenotypes. Numerous studies have successfully created Drosophila models for colorectal, lung, thyroid, and brain cancers. These models were utilized in the high-throughput screening of FDA-approved drugs which led to the identification of several compounds capable of reducing proliferation and rescuing phenotypes. More noteworthy, Drosophila has also unlocked the potential for personalized therapies. Drosophila 'avatars' presenting the same mutations as a patient are used to screen multiple therapeutic agents targeting multiple pathways to find the most appropriate combination of drugs. The outcomes of these studies have translated to significant responses in patients with adenoid cystic carcinoma and metastatic colorectal cancers. Despite not being widely utilized, the concept of in vivo screening of drugs in Drosophila is making significant contributions to the current drug discovery pipeline. In this review, we discuss the application of Drosophila as a platform in anticancer drug discovery; with special focus on the cancer models that have been generated, drug libraries that have been screened and the status of personalized therapies. In addition, we elaborate on the biological and technical limitations of this system.

The Tumor Microenvironment Factors That Promote Resistance to Immune Checkpoint Blockade Therapy.

Through genetic and epigenetic alterations, cancer cells present the immune system with a diversity of antigens or neoantigens, which the organism must distinguish from self. The immune system responds to neoantigens by activating naïve T cells, which mount an anticancer cytotoxic response. T cell activation begins when the T cell receptor (TCR) interacts with the antigen, which is displayed by the major histocompatibility complex (MHC) on antigen-presenting cells (APCs). Subsequently, accessory stimulatory or inhibitory molecules transduce a secondary signal in concert with the TCR/antigen mediated stimulus. These molecules serve to modulate the activation signal's strength at the immune synapse. Therefore, the activation signal's optimum amplitude is maintained by a balance between the costimulatory and inhibitory signals. This system comprises the so-called immune checkpoints such as the programmed cell death (PD-1) and Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and is crucial for the maintenance of self-tolerance. Cancers often evade the intrinsic anti-tumor activity present in normal physiology primarily by the downregulation of T cell activation. The blockade of the immune checkpoint inhibitors using specific monoclonal antibodies has emerged as a potentially powerful anticancer therapy strategy. Several drugs have been approved mainly for solid tumors. However, it has emerged that there are innate and acquired mechanisms by which resistance is developed against these therapies. Some of these are tumor-intrinsic mechanisms, while others are tumor-extrinsic whereby the microenvironment may have innate or acquired resistance to checkpoint inhibitors. This review article will examine mechanisms by which resistance is mounted against immune checkpoint inhibitors focussing on anti-CTL4-A and anti-PD-1/PD-Ll since drugs targeting these checkpoints are the most developed.

Solubilisation and purification of recombinant bluetongue virus VP7 expressed in a bacterial system.

Bluetongue virus (BTV) is an Orbivirus that has a profound economic impact due to direct loss of livestock as well as movement bans in an attempt to prevent the spread of the disease to susceptible areas. BTV VP7, along with VP3, forms the inner capsid core of the virus where it acts as the barrier between the outer layer and the inner core housing the genetic material. Purification of BTV VP7 has proven to be problematic and expensive mainly due to its insolubility is several expression systems. To overcome this, in this paper we present a protocol for the solubilisation of BTV VP7 from inclusion bodies expressed in E.coli, and subsequent purification using nickel affinity chromatography. The purified protein was then characterised using native PAGE, far ultraviolet circular dichroism (far-UV CD) and intrinsic fluorescence and found to have both secondary and tertiary structure even in the presence of 5 M urea. Both tertiary and secondary structure was further shown to be to be maintained at least to 42 °C in 5 M urea.

Analysis of Conserved, Computationally Predicted Epitope Regions for VP5 and VP7 Across three Orbiviruses.

Orbiviruses are double-stranded RNA viruses that have profound economic and veterinary significance, 3 of the most important being African horse sickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV). Currently, vaccination and vector control are used as preventative measures; however, there are several problems with the current vaccines. Comparing viral amino acid sequences, we obtained an AHSV-BTV-EHDV consensus sequence for VP5 (viral protein 5) and for VP7 (viral protein 7) and generated homology models for these proteins. The structures and sequences were analyzed for amino acid sequence conservation, entropy, surface accessibility, and epitope propensity, to computationally determine whether consensus sequences still possess potential epitope regions. In total, 5 potential linear epitope regions on VP5 and 11 on VP7, as well as potential discontinuous B-cell epitopes, were identified and mapped onto the homology models created. Regions identified for VP5 and VP7 could be important in vaccine design against orbiviruses.