Directed Evolution of Protease Beacons that Enable Sensitive Detection of Endogenous MT1-MMP Activity in Tumor Cell Lines

SummaryDirected evolution was applied to identify peptide substrates with enhanced hydrolysis rates by MT1-MMP suitable for protease beacon development. Screening of a random pentapeptide library, using two-color CLiPS, yielded several substrates identical to motifs in distinct collagens that shared the consensus sequence P-x-G↓L. To identify substrates with enhanced cleavage rates, a second-generation decapeptide library incorporating the consensus was screened under stringent conditions, which resulted in a MxPLG↓M/LMG/AR consensus motif. These substrates are hydrolyzed by human-MT1-MMP up to six times faster than reported peptide substrates and are stable in plasma. Finally, incubation of soluble protease beacons incorporating the optimized substrates, but not previous substrates, enabled direct detection of endogenous MT1-MMP activity of human-fibrosarcoma (HT-1080) cells. Extended substrate libraries coupled with CLiPS should be useful to generate more effective activity probes for a variety of proteolytic enzymes

Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library

The utility of blue fluorescent protein (BFP) has been limited by its low quantum yield and rapid photobleaching. A library targeting residues neighboring the chromophore yielded a variant with enhanced quantum yield (0.55 versus 0.34), reduced pH sensitivity and a 40-fold increase in photobleaching half-life. This BFP, named Azurite, is well expressed in bacterial and mammalian cells and extends the palette of fluorescent proteins that can be used for imaging

Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design

A host receptor enables type 1 pilus-mediated pathogenesis of Escherichia coli pyelonephritis

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis

An animal-specific FSI model of the abdominal aorta in anesthetized mice

Recent research has revealed that angiotensin II-induced abdominal aortic aneurysm in mice can be related to medial ruptures occurring in the vicinity of abdominal side branches. Nevertheless a thorough understanding of the biomechanics near abdominal side branches in mice is lacking. In the current work we present a mouse-specific fluid-structure interaction (FSI) model of the abdominal aorta in ApoE(-/-) mice that incorporates in vivo stresses. The aortic geometry was based on contrast-enhanced in vivo micro-CT images, while aortic flow boundary conditions and material model parameters were based on in vivo high-frequency ultrasound. Flow waveforms predicted by FSI simulations corresponded better to in vivo measurements than those from CFD simulations. Peak-systolic principal stresses at the inner and outer aortic wall were locally increased caudal to the celiac and left lateral to the celiac and mesenteric arteries. Interestingly, these were also the locations at which a tear in the tunica media had been observed in previous work on angiotensin II-infused mice. Our preliminary results therefore suggest that local biomechanics play an important role in the pathophysiology of branch-related ruptures in angiotensin-II infused mice. More elaborate follow-up research is needed to demonstrate the role of biomechanics and mechanobiology in a longitudinal setting

Connecting Health and Technology (CHAT): protocol of a randomized controlled trial to improve nutrition behaviours using mobile devices and tailored text messaging in young adults

Background: Increasing intakes of fruits and vegetables intake, in tandem with reducing consumption of energy-dense and nutrient poor foods and beverages are dietary priorities to prevent chronic disease. Although most adults do not eat enough fruit and vegetables, teenagers and young adults tend to have the lowest intakes. Young adults typically consume a diet which is inconsistent with the dietary recommendations. Yet little is known about the best approaches to improve dietary intakes and behaviours among this group. This randomised controlled trial aims to evaluate the effectiveness of using a mobile device to assess dietary intake, provide tailored dietary feedback and text messages to motivate changes in fruit, vegetable and junk food consumption among young adults

Evolution records a Mx tape for anti-viral immunity

Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx) genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host-virus interactions and species-specific susceptibility to viral infection

Author Correction: Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation