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Because rare, but severe adverse effects, i.e. retinal vasculitis or retinal vein occlusion, have been observed after repetitive intravitreal injections of VEGF-A-binding single-chain variable fragment brolucizumab (Beovu), we investigated its possible impact on the barrier formed by immortalized bovine retinal endothelial cells (iBREC) in comparison to that of the VEGF-A-binding Fab fragment ranibizumab (Lucentis). As a measure of stability of the barrier formed by a confluent monolayer of iBREC, we determined the cell index over seven days by continuous electric cell-substrate impedance measurements: Beovu but not Lucentis indeed significantly lowered the cell index, evident about 1.5 days after its addition, pointing to barrier impairment. Early after addition of Beovu, amounts of the integrins α5 and β1—subunits of the fibronectin receptor—had changed in opposite ways, suggesting an effect on cell adhesion due to hindered dimer formation. After exposure for eight days to Beovu, levels of claudin-1—an essential part of the iBREC barrier—were significantly lower, less claudin-1 was located at the plasma membrane after exposure to the VEGF-A antagonist for five days. Beovu did not induce secretion of inflammatory cytokines or VEGF-A. Interestingly, polysorbate-80—component of Beovu—but not polysorbate-20—in Lucentis—slightly, but significantly lowered the cell index, also associated with reduced claudin-1 expression. In summary, our results indicate that Beovu changes the behavior of retinal endothelial cells, thus providing an alternative “non-immunological” explanation for the most relevant of observed side effects.

Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo -inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS.

The beneficial effects of Cyclooxygenases (COX) inhibitors on human health have been known for thousands of years. Nevertheless, COXs, particularly COX-1, have been linked to a plethora of human diseases such as cancer, heart failure, neurological and neurodegenerative diseases only recently. COXs catalyze the first step in the biosynthesis of prostaglandins (PGs) and are among the most important mediators of inflammation. All published structural work on COX-1 deals with the ovine isoenzyme, which is easier to produce in milligram-quantities than the human enzyme and crystallizes readily. Here, we report the long-sought structure of the human cyclooxygenase-1 ( h COX-1) that we refined to an R/R free of 20.82/26.37, at 3.36 Å resolution. h COX-1 structure provides a detailed picture of the enzyme active site and the residues crucial for inhibitor/substrate binding and catalytic activity. We compared h COX-1 crystal structure with the ovine COX-1 and human COX-2 structures by using metrics based on Cartesian coordinates, backbone dihedral angles, and solvent accessibility coupled with multivariate methods. Differences and similarities among structures are discussed, with emphasis on the motifs responsible for the diversification of the various enzymes (primary structure, stability, catalytic activity, and specificity). The structure of h COX-1 represents an essential step towards the development of new and more selective COX-1 inhibitors of enhanced therapeutic potential.

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon’s N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon’s NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon’s activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon’s importance in maintaining proteostasis in mitochondria.

Invasive fungal infections mainly affect patients undergoing transplantation, surgery, neoplastic disease, immunocompromised subjects and premature infants, and cause over 1.5 million deaths every year. The most common fungi isolated in invasive diseases are Candida spp ., Cryptococcus spp ., and Aspergillus spp. and even if four classes of antifungals are available (Azoles, Echinocandins, Polyenes and Pyrimidine analogues), the side effects of drugs and fungal acquired and innate resistance represent the major hurdles to be overcome. Monoclonal antibodies are powerful tools currently used as diagnostic and therapeutic agents in different clinical contexts but not yet developed for the treatment of invasive fungal infections. In this paper we report the development of the first humanized monoclonal antibody specific for β-1,3 glucans, a vital component of several pathogenic fungi. H5K1 has been tested on C . auris, one of the most urgent threats and resulted efficient both alone and in combination with Caspofungin and Amphotericin B showing an enhancement effect. Our results support further preclinical and clinical developments for the use of H5K1 in the treatment of patients in need.

The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels. The production conditions were optimized and EfmRseP was purified by immobilized metal ion affinity chromatography followed by size-exclusion chromatography. The purification resulted in an overall yield of approximately 1 mg of pure protein per 3 g of wet-weight cell pellet. The structural integrity of the purified protein was confirmed using circular dichroism. We further assessed the expression and purification of RseP from E. faecium in the Gram-negative Escherichia coli . Detection of soluble protein failed in two of the three E. coli strains tested. Purification of EfmRseP expressed in E. coli C43(DE3) resulted in a protein with lower purity compared to EfmRseP expressed in L. plantarum . To our knowledge, this is the first time L. plantarum and the pSIP expression system have been applied for the production of membrane proteins.

A critical step in proteomics analysis is the optimal extraction and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technology that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-preparation (SP3) technology is a paramagnetic bead–based approach for rapid, robust, and efficient processing of protein samples for proteomic analysis. SP3 uses a hydrophilic interaction mechanism for exchange or removal of components that are commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymatic digestion (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) before downstream proteomic analysis. The SP3 protocol consists of nonselective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads, and elution of purified material in aqueous conditions. In contrast to alternative approaches, SP3 combines compatibility with a substantial collection of solution additives with virtually lossless and unbiased recovery of proteins independent of input quantity, all in a simplified single-tube protocol. The SP3 protocol is simple and efficient, and can be easily completed by a standard user in ~30 min, including reagent preparation. As a result of these properties, SP3 has successfully been used to facilitate examination of a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms. This work describes the steps and extensive considerations involved in performing SP3 in bottom-up proteomics, using a simplified protein cleanup scenario for illustration.

The ability to measure plant hormones quantitatively is important as plant hormones regulate plant growth, development and response to biotic and abiotic cues. In this protocol, we describe the quantitative analysis of major plant hormones from crude plant extracts. Plant hormones are determined using reverse-phase liquid chromatography–tandem mass spectrometry with multiple reaction monitoring. The method provides quantification of most major plant hormones in a single run from 50 mg of fresh plant tissue. Extraction and quantitative analysis of 40 samples takes 2–3 d.

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable and effective method to purify EVs taking advantage of their heparin affinity.

Crop plants contain large amounts of secondary compounds that interfere with protein extraction and gel-based proteomic analysis. Thus, a protein extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and phenol extraction. Through SDS extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by phenol extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and phenol extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.