Using Protein Engineering to Understand and Modulate Aggregation

Protein aggregation occurs through a variety of mechanisms, initiated by the unfolded, non-native, or even the native state itself. Understanding the molecular mechanisms of protein aggregation is challenging, given the array of competing interactions that control solubility, stability, cooperativity and aggregation propensity. An array of methods have been developed to interrogate protein aggregation, spanning computational algorithms able to identify aggregation-prone regions, to deep mutational scanning to define the entire mutational landscape of a protein’s sequence. Here, we review recent advances in this exciting and emerging field, focussing on protein engineering approaches that, together with improved computational methods, hold promise to predict and control protein aggregation linked to human disease, as well as facilitating the manufacture of protein-based therapeutics

Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules

Protein–protein interactions (PPIs) are involved in many of life’s essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein β2-microglobulin (β2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein–fragment interaction. The covalent protein–ligand linkage enabled structural characterization of these trapped, off-pathway oligomers using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of “post-translational chemical modification” as a tool to study biological molecular mechanisms

An in vivo platform to select and evolve aggregation-resistant proteins

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve ‘manufacturable’ biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease

Prescribing practices of primary-care veterinary practitioners in dogs diagnosed with bacterial pyoderma

Concern has been raised regarding the potential contributions of veterinary antimicrobial use to increasing levels of resistance in bacteria critically important to human health. Canine pyoderma is a frequent, often recurrent diagnosis in pet dogs, usually attributable to secondary bacterial infection of the skin. Lesions can range in severity based on the location, total area and depth of tissue affected and antimicrobial therapy is recommended for resolution. This study aimed to describe patient signalment, disease characteristics and treatment prescribed in a large number of UK, primary-care canine pyoderma cases and to estimate pyoderma prevalence in the UK vet-visiting canine population

Down-regulation of TM4SF is associated with the metastatic potential of gastric carcinoma TM4SF members in gastric carcinoma

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to clarify the clinical significance of TM4SF members CD9, CD63 and CD82 in human gastric carcinoma.</p> <p>Methods</p> <p>By employing RT-PCR and immunohistochemistry, we studied the expression of CD9, CD63 and CD82 in 49 paired tissue specimens of normal gastric mucosa and carcinoma. All tissues were obtained from patients who underwent curative surgery.</p> <p>Results</p> <p>All normal gastric epithelium and gastric ulcer tissues strongly expressed transcripts and proteins of CD9, CD63 and CD82 as compared with corresponding controls. We found a significant correlation between CD63 mRNA level and different pM statuses (P = 0.036). Carcinomas in M0 stage revealed a stronger expression of CD63 than carcinomas in M1 stage. Expression of CD9 protein was found significantly stronger in pN0, pM0 than in advanced pN stages (P = 0.03), pM1 (P = 0.013), respectively. We found the relationship between CD63 expression, gender (p = 0.09) and nodal status (p = 0.028), respectively. Additionally, advanced and metastasized tumor tissues revealed significantly down-regulated CD82 protein expression (p = 0.033 and p = 0, respectively), which correlated with the tumor pTNM stage (p = 0.001).</p> <p>Conclusion</p> <p>The reduction of CD9, CD63 and CD82 expression are indicators for the metastatic potential of gastric carcinoma cells. Unlike their expression in other tumor types, the constitutive expression of CD63 may indicate that this factor does play a direct role in human gastric carcinogenesis.</p

Phase 1b study of tirabrutinib in combination with idelalisib or entospletinib in previously treated B-cell lymphoma.

B-cell receptor (BCR) signaling pathway inhibitors (including Bruton’s tyrosine kinase [BTK] inhibitors, and phosphatidylinositol-3 kinase inhibitors [PI3Ki]) have shown clinical efficacy in non-Hodgkin lymphoma (NHL). However, responses to these agents have been limited in depth and duration. This may be due to resistance to PI3Kδ and BTK inhibitors as monotherapy. The emergence of resistant clones may be addressed by combining these 2 classes of drugs. Furthermore, tolerability of these drug classes has been a concern. Combination therapy using lower doses of one or more classes of inhibitor may address some limitations

Cup Blocks the Precocious Activation of the Orb Autoregulatory Loop

Translational regulation of localized mRNAs is essential for patterning and axes determination in many organisms. In the Drosophila ovary, the germline-specific Orb protein mediates the translational activation of a variety of mRNAs localized in the oocyte. One of the Orb target mRNAs is orb itself, and this autoregulatory activity ensures that Orb proteins specifically accumulate in the developing oocyte. Orb is an RNA-binding protein and is a member of the cytoplasmic polyadenylation element binding (CPEB) protein family. We report here that Cup forms a complex in vivo with Orb. We also show that cup negatively regulates orb and is required to block the precocious activation of the orb positive autoregulatory loop. In cup mutant ovaries, high levels of Orb accumulate in the nurse cells, leading to what appears to be a failure in oocyte specification as a number of oocyte markers inappropriately accumulate in nurse cells. In addition, while orb mRNA is mislocalized and destabilized, a longer poly(A) tail is maintained than in wild type ovaries. Analysis of Orb phosphoisoforms reveals that loss of cup leads to the accumulation of hyperphosphorylated Orb, suggesting that an important function of cup in orb-dependent mRNA localization pathways is to impede Orb activation

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

The Sir Jimmy Savile Scandal: Child Sexual Abuse and Institutional Denial at the BBC

This study advances research on scandal through an empirical examination of one of the most extraordinary UK institutional child sexual abuse (CSA) scandals in the post-war period. Sir Jimmy Savile (1926–2011) was a BBC celebrity, showbiz friend of the establishment and philanthropist. In October 2012, one year after his death, an ITV documentary alleged that Savile was also a prolific sexual predator who for decades had exploited his BBC status to abuse teenage girls. As we demonstrate, this incendiary documentary triggered a news media feeding frenzy that in less than one week destroyed Savile’s reputation and thrust the BBC – the institution that made him a star – into a multi-faceted, globally reported CSA scandal. This study has four purposes. First, we propose a model of institutional CSA scandals that can account for critical transitions between key phases in the scandal process. Second, we apply this model to analyse the transition between the ‘latent’ and ‘activated’ phases of the Savile scandal. This transition corresponded with a dramatic transformation in the inferential structuring of Savile from ‘national treasure’, who had devoted decades to working with children, to ‘prolific sexual predator’, who spent decades abusing them. Third, we demonstrate how the BBC’s denial of responsibility for Savile’s sexual offending and its subsequent institutional cover-up triggered a ‘trial by media’ which in turn initiated the next phase in the scandal’s development – ‘amplification’. Finally, we consider the significance of our analysis of the Sir Jimmy Savile scandal for understanding the activation and development of scandals more generally

Variation at GRN 3′-UTR rs5848 Is Not Associated with a Risk of Frontotemporal Lobar Degeneration in Dutch Population

Background: A single nucleotide polymorphism (rs5848) located in the 3′- untranslated region of GRN has recently been associated with a risk of frontotemporal lobar degeneration (FTLD) in North American population particularly in pathologically confirmed cases with neural inclusions immunoreactive for ubiquitin and TAR DNA-binding protein 43 (TDP-43), but negative for tau and alpha-synuclein (FTLD-TDP). Methodology/Principal Findings: In an effort to replicate these results in a different population, rs5848 was genotyped in 256 FTLD cases and 1695 controls from the Netherlands. Single SNP gender-adjusted logistic regression analysis revealed no significant association between variation at rs5848 and FTLD. Fisher's exact test, failed to find any significant association between rs5848 and a subset of 23 pathology confirmed FTLD-TDP cases. Conclusions/Significance: The evidence presented here suggests that variation at rs5848 does not contribute to the etiology of FTLD in the Dutch population