Efficient protein depletion by genetically controlled deprotection of a dormant N-degron

Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology

Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease

AbstractBinding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential

Performance of artificial intelligence for colonoscopy regarding adenoma and polyp detection: a meta-analysis

BACKGROUND AND AIMS One fourth of colorectal neoplasia is missed at screening colonoscopy, representing the main cause of interval colorectal cancer (CRC). Deep learning systems with real-time computer-aided polyp detection (CADe) showed high accuracy in artificial settings, and preliminary randomized clinical trials (RCT) reported favourable outcomes in clinical setting. Aim of this meta-analysis was to summarise available RCTs on the performance of CADe systems in colorectal neoplasia detection. METHODS We searched MEDLINE, EMBASE and Cochrane Central databases until March 2020 for RCTs reporting diagnostic accuracy of CADe systems in detection of colorectal neoplasia. Primary outcome was pooled adenoma detection rate (ADR), Secondary outcomes were adenoma per colonoscopy (APC) according to size, morphology and location, advanced APC (AAPC), as well as polyp detection rate (PDR), Polyp-per-colonoscopy (PPC), and sessile serrated lesion per colonoscopy (SPC). We calculated risk ratios (RR), performed subgroup, and sensitivity analysis, assessed heterogeneity, and publication bias. RESULTS Overall, 5 randomized controlled trials (4354 patients), were included in the final analysis. Pooled ADR was significantly higher in the CADe groups than in the control group (791/2163, 36.6% vs 558/2191, 25.2%; RR, 1.44; 95% CI, 1.27-1.62; p10 mm adenomas (RR, 1.46; 95% CI, 1.04-2.06), as well as for proximal (RR, 1.59; 95% CI, 1.34-1.88) and distal (RR, 1.68; 95% CI, 1.50-1.88), and for flat (RR: 1.78 95% CI 1.47-2.15) and polypoid morphology (RR, 1.54; 95% CI, 1.40-1.68). Regarding histology, CADe resulted in a higher SPC (RR, 1.52; 95% CI,1.14-2.02), whereas a nonsignificant trend for AADR was found (RR, 1.35; 95% CI, 0.74 – 2.47; p = 0.33; I 2:69%). Level of evidence for RCTs was graded moderate. CONCLUSIONS According to available evidence, the incorporation of Artificial Intelligence as aid for detection of colorectal neoplasia results in a significant increase of the detection of colorectal neoplasia, and such effect is independent from main adenoma characteristics

Prophylactic Clipping After Colorectal Endoscopic Resection Prevents Bleeding of Large, Proximal Polyps: Meta-Analysis of Randomized Trials

Background & Aims The benefits of prophylactic clipping to prevent bleeding after polypectomy are unclear. We conducted an updated meta-analysis of randomized trials to assess the efficacy of clipping in preventing bleeding after polypectomy, overall and according to polyp size and location. Methods We searched the Medline/PubMed, EMBASE, and Scopus databases randomized trials that compared effects of clipping vs not clipping to prevent bleeding after polypectomy. We performed a random-effects meta-analysis to generate pooled relative risks (RRs) with 95% CIs. Multilevel random-effects meta-regression analysis was used to combine data on bleeding after polypectomy and estimate associations between rates of bleeding and polyp characteristics. Results We analyzed data from 9 trials, comprising 7197 colorectal lesions (22.5% 20 mm or larger, 49.2% with proximal location). Clipping, compared with no clipping, did not significantly reduce the overall risk of post-polypectomy bleeding (2.2% with clipping vs 3.3% with no clipping; RR, 0.69; 95% CI, 0.45–1.08; P=.072). Clipping significantly reduced risk of bleeding after removal of polyps that were 20 mm or larger (4.3% had bleeding after clipping vs 7.6% had bleeding with no clipping; RR, 0.51; 95% CI, 0.33–0.78; P=.020) or that were in a proximal location (3.0% had bleeding after clipping vs 6.2% had bleeding with no clipping; RR, 0.53; 95% CI, 0.35–0.81; P<.001). In multilevel meta-regression analysis that adjusted for polyp size and location, prophylactic clipping was significantly associated with reduced risk of bleeding after removal of large proximal polyps (RR, 0.37; 95% CI, 0.22–0.61; P=.021) but not small proximal lesions (RR, 0.88; 95% CI, 0.48–1.62; P=0.581). Conclusions In a meta-analysis of randomized trials, we found that routine use of prophylactic clipping does not reduce risk of post-polypectomy bleeding, overall. However, clipping appeared to reduce bleeding after removal of large (more than 20 mm), proximal lesions

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges

A tobacco etch virus protease with increased substrate tolerance at the P1' position.

Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein-protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1' Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1' Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid

Plasmids used in this study.

<p>Plasmids used in this study.</p

Generation of a TEV protease that cleaves efficiently the recognition sequence ENLYFQ-R.

<p><b>A</b>) Scheme of the construct used for the screening procedure: The bidirectional degron module GFP-cODC1-TDegX-RFP (X = F or R) was fused to the phosphoribosylaminoimidazole carboxylase Ade2. Cleavage by the TEV protease leads to activation of the C-degron cODC1 and the N-degron TDegX resulting in proteasomal degradation of Ade2-GFP-cODC1 as well as TDegX-RFP. <b>B</b>) Test for adenine biosynthesis in cells bearing different degron constructs fused chromosomally to <i>ADE2</i>. The yeast strains (ESM356-1, YCT1266, and YCR8) were grown in patches on solid media (YPD, YP+galactose, yeast nitrogen base + 2% glucose, and yeast nitrogen base + 2% galactose; from left to right). <b>C</b>) Scheme illustrating the mutagenesis and selection procedure to obtain a TEV protease which efficiently processes the recognition sequence ENLYFQ-R (left side). The plate is an example to show the difference in color of clones with efficient proteolysis of ENLYFQ-R (red colonies) and clones with insufficient proteolysis (white colonies). Please note that the high degree of red colonies was obtained because the R345G mutant was generated already in the first round of mutagenesis and enriched in subsequent rounds. <b>D</b>) Expression of <i>pTEV2 protease</i> (plasmid-based, R345G mutant) using the <i>GAL1</i> promoter induces the adenine auxotrophy phenotype in <i>ade2-GFP-cODC1-TDegR-RFP</i> cells (YCR6). Serial dilutions (1:10) were grown on solid media as in B.</p