Inhibition of prolyl oligopeptidase : A promising pathway to prevent the progression of age-related macular degeneration

Dry age-related macular degeneration (AMD) is a currently untreatable vision threatening disease. Impaired proteasomal clearance and autophagy in the retinal pigment epithelium (RPE) and subsequent photoreceptor damage are connected with dry AMD, but detailed pathophysiology is still unclear. In this paper, we discover inhibition of cytosolic protease, prolyl oligopeptidase (PREP), as a potential pathway to treat dry AMD. We showed that PREP inhibitor exposure induced autophagy in the RPE cells, shown by increased LC3-II levels and decreased p62 levels. PREP inhibitor treatment increased total levels of autophagic vacuoles in the RPE cells. Global proteomics was used to examine the phenotype of a commonly used cell model displaying AMD characteristics, oxidative stress and altered protein metabolism, in vitro. These RPE cells displayed induced protein aggregation and clear alterations in macromolecule metabolism, confirming the relevance of the cell model. Differences in intracellular target engagement of PREP inhibitors were observed with cellular thermal shift assay (CETSA). These differences were explained by intracellular drug exposure (the unbound cellular partition coefficient, Kpuu). Importantly, our data is in line with previous observations regarding the discrepancy between PREP's cleaving activity and outcomes in autophagy. This highlights the need to further explore PREP's role in autophagy so that more effective compounds can be designed to battle diseases in which autophagy induction is needed. The present work is the first report investigating the PREP pathway in the RPE and we predict that the PREP inhibitors can be further optimized for treatment of dry AMD.Peer reviewe

Identification of new regulatory mechanisms that determine coagulation FXI plasma concentration

FXI is a protein in the coagulation cascade in humans proven to be involved in the propagation and stabilization of developing thrombi in previous studies. The regulatory mechanisms for FXI are not well understood. Therefore an investigation to find regulatory mechanisms for FXI was performed. A meta-analysis was conducted, consisting of six Genome-wide association studies in which the trait was FXI levels in plasma. Three genome wide significant loci were found that also were significant in a replication study, in which three cohorts not included in the discovery set were used. Functional annotation, pathway analysis and eQTL analysis of these three loci yielded three genes believed potentially responsible for the regulation of FXI. Two of these three genes were chosen for a microRNA binding prediction search. Several microRNAs were found, and one was analyzed with a luciferase reporter assa

Proteomics informed investigation of human hepatocytes and liver tissue

A successful drug needs to display beneficial absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) profile. It is therefore important to investigate these properties during the drug discovery process. The liver is of particular interest in ADME-Tox studies, as it is highly metabolically active and oral administrated drugs needs to pass the liver before reaching the systemic circulation. However, a dose of a drug that is efficacious and safe for one individual may be inefficacious or toxic, because of inter-individual variability. Therefore, it is important to investigate the ADME-Tox properties in a sufficiently large population. Investigations on ADME-Tox is usually done in in vitro cell models.  Therefore, a variety of models to simulate liver functions have been developed and ranging from subcellular microsomes to complex 3D organoid cultures. This thesis investigates variability of ADME proteins in human liver tissue and in liver cell models. First, mass spectrometry based targeted proteomics was used to quantify ADME relevant proteins from 149 human liver samples. The observed inter-individual protein variability could not solely be explained by genotype. Therefore, a single transporter protein, the bile and drug transporting protein, NTCP, was investigate in detail.  Non-genetic factors, e.g. smoking and alcohol consumption, and epigenetic factors such as DNA methylation, were found to contribute to the observed inter-individual variability of NTCP.  Next, hepatocytes (PHH) were isolated from 54 human livers tissues and after which the hepatocytes where cryopreserved. The variable attachment efficiency of cryopreserved hepatocytes where investigated and an apoptosis inhibition protocol for restoration of attachment properties was developed. This protocol was also successfully applied to 3D cultured PHH spheroids resulting in increased ability to form 3D spheroids.  The effect of culture conditions on the quality of the 3D cultures was also investigated.  3D PHH spheroids were formed and maintained in different, commonly used culture media. The spheroids were characterized by a variety of functional assays including global proteomics. The proteome analysis showed that while no epithelial to mesenchymal transitions was observed, 3D cultures maintained in fasting glucose and insulin levels resembling the in vivo situation showed a more liver-like phenotype with a high expression of ADME proteins and functional cytochrome P450 metabolism. Transporter kinetics were also investigated in the 3D cultured PHH.  Finally, we investigated if global proteomics data from 56 human liver tissues could be deconvoluted to give information about the liver composition. The cell type proportions generated by deconvolution where similar to literature values. Liver samples that displayed deviating cell composition were identified. The deviating liver compositions were in agreement with clinical markers of inflammation in the patient´s blood samples and with altered extracellular matrix protein composition, comparable to that found in liver steatosis.   In conclusion, this thesis have investigated variability in ADME proteins in human liver and in in vitro cultures of human hepatocytes, characterized cofounding factors for in vitro cultured hepatocytes and further extended drug disposition studies in 3D cultured hepatocytes. https://doi.org/10.33063/diva-446954</p

Image-based quantification of cell debris as a measure of apoptosis

Apoptosis is a controlled form of cell death that can be induced by various diseases and exogenous toxicants. Common apoptosis detection methods rely on fluorescent markers, which necessitates the use of costly reagents and time-consuming labeling procedures. Label-free methods avoid these problems, but often require specialized instruments instead. Here, we utilize apoptotic cell disintegration to develop a novel label-free detection method based on the quantification of subcellular debris particles in bright-field microscopy images. Debris counts show strong correlations with fluorescence-based annexin V staining, and can be used to study concentration-dependent and temporal apoptosis activation. The method is rapid, low-cost, and easy to apply, as the only experimental step comprises bright-field imaging of culture media samples, followed by automated image processing. The late-stage nature of the debris measurement means that the method can complement other, established apoptosis assays, and its accessibility will allow a wider community of researchers to study apoptotic cell death

Mot en effektiv data- och informationshantering på SiCell

Denna projektrapport är avsedd att vara ett hjälpmedel för SiCell, en del av SciLifeLab Uppsala som ska bli Europas första plattform för enkelcellgenomik till hösten 2013. SiCell har bett projektgruppen om undersökningar gällande ett Laboratory Information Management System, LIMS. På svenska ett datahanteringsystem för laboratorier. Ett sådant system skulle kunna effektivisera SiCells verksamhet. Undersökningarna har resulterat i en kravspecifikation som ett LIMS för SiCell ska uppfylla och en sammanställning av tillgängliga mjukvaror som bäst uppfyller dessa krav. Screensaver, MISO och Gnomex är de tre gratisprogram med öppen källkod som hamnade högst upp i listan. Inget av dem uppfyller alla krav men med modifieringar av programmerare tros detta vara möjligt. SiCell bad också om lägre prioriterade undersökningar av några av de metoder som används inom plattformen. Cellysering, Alternativa amplifieringsmetoder och transkriptomik har undersökts av projektgruppen. Detta resulterade i en sammanställning av vilka alternativ som finns och vad som är under utveckling inom respektive område

Mot en effektiv data- och informationshantering på SiCell

Denna projektrapport är avsedd att vara ett hjälpmedel för SiCell, en del av SciLifeLab Uppsala som ska bli Europas första plattform för enkelcellgenomik till hösten 2013. SiCell har bett projektgruppen om undersökningar gällande ett Laboratory Information Management System, LIMS. På svenska ett datahanteringsystem för laboratorier. Ett sådant system skulle kunna effektivisera SiCells verksamhet. Undersökningarna har resulterat i en kravspecifikation som ett LIMS för SiCell ska uppfylla och en sammanställning av tillgängliga mjukvaror som bäst uppfyller dessa krav. Screensaver, MISO och Gnomex är de tre gratisprogram med öppen källkod som hamnade högst upp i listan. Inget av dem uppfyller alla krav men med modifieringar av programmerare tros detta vara möjligt. SiCell bad också om lägre prioriterade undersökningar av några av de metoder som används inom plattformen. Cellysering, Alternativa amplifieringsmetoder och transkriptomik har undersökts av projektgruppen. Detta resulterade i en sammanställning av vilka alternativ som finns och vad som är under utveckling inom respektive område

A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes

Primary human hepatocytes are used in all facets of liver research, from in vitro studies of xenobiotic disposition and toxicity to the clinical management of liver failure. Unfortunately, cellular stress during isolation and cryopreservation causes a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. Reasoning that this problem could be mitigated at the post-thawing stage, we applied label-free quantitative global proteomics to analyze differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, ultimately leading to apoptosis activation. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately post-thawing, which suggested the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. Brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored attachment ability and promoted a differentiated morphology, as well as formation of 3D spheroids. Further, Z-VAD-FMK treatment restored metabolic and transport functions, with maintained sensitivity to hepatotoxic insults. Altogether, this study shows that differentiation and function of suboptimal human hepatocytes can be restored after cryopreservation, thus markedly increasing the availability of these precious cells

Mot en effektiv data- och informationshantering på SiCell

Denna projektrapport är avsedd att vara ett hjälpmedel för SiCell, en del av SciLifeLab Uppsala som ska bli Europas första plattform för enkelcellgenomik till hösten 2013. SiCell har bett projektgruppen om undersökningar gällande ett Laboratory Information Management System, LIMS. På svenska ett datahanteringsystem för laboratorier. Ett sådant system skulle kunna effektivisera SiCells verksamhet. Undersökningarna har resulterat i en kravspecifikation som ett LIMS för SiCell ska uppfylla och en sammanställning av tillgängliga mjukvaror som bäst uppfyller dessa krav. Screensaver, MISO och Gnomex är de tre gratisprogram med öppen källkod som hamnade högst upp i listan. Inget av dem uppfyller alla krav men med modifieringar av programmerare tros detta vara möjligt. SiCell bad också om lägre prioriterade undersökningar av några av de metoder som används inom plattformen. Cellysering, Alternativa amplifieringsmetoder och transkriptomik har undersökts av projektgruppen. Detta resulterade i en sammanställning av vilka alternativ som finns och vad som är under utveckling inom respektive område

High Throughput Screening of a Prescription Drug Library for Inhibitors of Organic Cation Transporter 3, OCT3

Introduction The organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental toxins and prescription drugs. Understudied as a determinant of pharmacokinetics and pharmacodynamics, OCT3 has the potential to be a major determinant of drug absorption and disposition and to be a target for drug-drug interactions (DDIs). Goal The goal of the current study was to identify prescription drug inhibitors of OCT3. Methods We screened a compound library consisting of 2556 prescription drugs, bioactive molecules, and natural products using a high throughput assay in HEK-293 cells stably expressing OCT3. Results We identified 210 compounds that at 20 mu M inhibit 50% or more of OCT3-mediated uptake of 4-Di-1-ASP (2 mu M). Of these, nine were predicted to inhibit the transporter at clinically relevant unbound plasma concentrations. A Structure-Activity Relationship (SAR) model included molecular descriptors that could discriminate between inhibitors and non-inhibitors of OCT3 and was used to identify additional OCT3 inhibitors. Proteomics of human brain microvessels (BMVs) indicated that OCT3 is the highest expressed OCT in the human blood-brain barrier (BBB). Conclusions This study represents the largest screen to identify prescription drug inhibitors of OCT3. Several are sufficiently potent to inhibit the transporter at therapeutic unbound plasma levels, potentially leading to DDIs or off-target pharmacologic effects

Common and mutation specific phenotypes of KRAS and BRAF mutations in colorectal cancer cells revealed by integrative -omics analysis

Background: Genes in the Ras pathway have somatic mutations in at least 60 % of colorectal cancers. Despite activating the same pathway, the BRAF V600E mutation and the prevalent mutations in codon 12 and 13 of KRAS have all been linked to different clinical outcomes, but the molecular mechanisms behind these differences largely remain to be clarified. Methods: To characterize the similarities and differences between common activating KRAS mutations and between KRAS and BRAF mutations, we used genome editing to engineer KRAS G12C/D/V and G13D mutations in colorectal cancer cells that had their mutant BRAF V600E allele removed and subjected them to transcriptome sequencing, global proteomics and metabolomics analyses. Results: By intersecting differentially expressed genes, proteins and metabolites, we uncovered (i) two-fold more regulated genes and proteins when comparing KRAS to BRAF mutant cells to those lacking Ras pathway mutation, (ii) five differentially expressed proteins in KRAS mutants compared to cells lacking Ras pathway mutation (IFI16, S100A10, CD44, GLRX and AHNAK2) and 6 (CRABP2, FLNA, NXN, LCP1, S100A10 and S100A2) compared to BRAF mutant cells, (iii) 19 proteins expressed differentially in a KRAS mutation specific manner versus BRAF V600E cells, (iv) regulation of the Integrin Linked Kinase pathway by KRAS but not BRAF mutation, (v) regulation of amino acid metabolism, particularly of the tyrosine, histidine, arginine and proline pathways, the urea cycle and purine metabolism by Ras pathway mutations, (vi) increased free carnitine in KRAS and BRAF mutant RKO cells. Conclusions: This comprehensive integrative -omics analysis confirms known and adds novel genes, proteins and metabolic pathways regulated by mutant KRAS and BRAF signaling in colorectal cancer. The results from the new model systems presented here can inform future development of diagnostic and therapeutic approaches targeting tumors with KRAS and BRAF mutations