Publications by authors named "Jaco Knol"

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity.

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Article Synopsis
  • The study focuses on abdominal aortic aneurysm (AAA), a condition where the aorta weakens and dilates in the abdomen, aiming to understand the specific cellular mechanisms involved by analyzing proteins in vascular smooth muscle cells (VSMC) from AAA patients versus healthy donors.
  • Using advanced proteomic techniques, researchers found significant differences in proteins linked to extracellular matrix remodeling, energy metabolism, and muscle contractility between AAA patients and healthy individuals.
  • The research revealed changes in phosphorylation patterns affecting structural proteins like those involved in the actin cytoskeleton and signaling pathways, suggesting specific kinases like NUAK1 and MAPK7 may play crucial roles in AAA development.
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Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors.

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The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment with high detectability in biofluids. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: (1) Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); (2) Acetone protein precipitation coupled to IGD; (3) MWCO filter-based protein concentration followed by to in-solution digestion (ISD); (4) Acetone protein precipitation coupled to ISD; (5) Direct ISD; (6) Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines, and growth factors.

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The tyrosine kinase inhibitor sunitinib is an effective first-line treatment for patients with advanced renal cell carcinoma (RCC). Hypothesizing that a functional read-out by mass spectrometry-based (phospho, p-)proteomics will identify predictive biomarkers for treatment outcome of sunitinib, tumor tissues of 26 RCC patients were analyzed. Eight patients had primary resistant (RES) and 18 sensitive (SENS) RCC.

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Background And Aim: Phenotypic expression of hypertrophic cardiomyopathy (HCM) and disease course are associated with unfavorable metabolic health. We investigated if Western diet (WD) feeding is sufficient to trigger cardiac hypertrophy and dysfunction in heterozygous (HET) knock-in mice.

Methods And Results: Wild-type (WT) and HET mice (3-months-old) were fed a WD or normal chow (NC) for 8 weeks.

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Proteogenomic analysis is emerging as an advantageous tool to assist personalized therapy decisions in clinical health care and integrates complementary information from the genome, transcriptome, and (phospho)proteome. A prerequisite for such analysis is a workflow for the simultaneous isolation of DNA, RNA, and protein from a single sample that does not compromise the different biological molecules and their examination. Focusing on the phosphoproteomic aspect of this workflow, we here provide detailed information on our protocol, which is based on commonly used acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction with RNA-Bee.

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Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry-based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient-derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance.

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Background: Epicardial adipose tissue (EAT) secretome induces fibrosis. Fibrosis, primarily extracellular matrix (ECM) produced by fibroblasts, creates a substrate for atrial fibrillation (AF). Whether the EAT secretome from patients with AF activates human atrial fibroblasts and through which components, remains unexplored.

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Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited set of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a readout of aberrant signaling and has the potential to identify new targets and guide treatment decisions. Using two-step sequential phosphopeptide enrichment, we generate a comprehensive phosphoproteome and proteome of nine PDAC cell lines, encompassing more than 20,000 phosphosites on 5,763 phospho-proteins, including 316 protein kinases.

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Introduction: Extracellular vesicles (EVs) may propagate and modulate Alzheimer's disease (AD) pathology. We aimed to comprehensively characterize the proteome of cerebrospinal fluid (CSF) EVs to identify proteins and pathways altered in AD.

Methods: CSF EVs were isolated by ultracentrifugation (Cohort 1) or Vn96 peptide (Cohort 2) from non-neurodegenerative controls (n = 15, 16) and AD patients (n = 22, 20, respectively).

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Purpose: In precision oncology, tumor molecular profiles guide selection of therapy. Standardized snap freezing of tissue biospecimens is necessary to ensure reproducible, high-quality samples that preserve tumor biology for adequate molecular profiling. Quenching in liquid nitrogen (LN ) is the golden standard method, but LN has several limitations.

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In Birt-Hogg-Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCN and FLCN human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins.

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Purpose: Tyrosine kinase inhibitors (TKI) have poor efficacy in patients with glioblastoma (GBM). Here, we studied whether this is predominantly due to restricted blood-brain barrier penetration or more to biological characteristics of GBM.

Patients And Methods: Tumor drug concentrations of the TKI sunitinib after 2 weeks of preoperative treatment was determined in 5 patients with GBM and compared with its in vitro inhibitory concentration (IC50) in GBM cell lines.

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Platelets are involved in tumor angiogenesis and cancer progression. Previous studies indicated that cancer could affect platelet content. In the current study, we investigated whether cancer-associated proteins can be discerned in the platelets of cancer patients, and whether antitumor treatment may affect the platelet proteome.

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Sex-differences in clinical presentation contribute to the phenotypic heterogeneity of hypertrophic cardiomyopathy (HCM) patients. While disease prevalence is higher in men, women present with more severe diastolic dysfunction and worse survival. Until today, little is known about the cellular differences underlying sex-differences in clinical presentation.

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Purpose: To mimic the perioperative microenvironment where bacterial products get in contact with colorectal cancer (CRC) cells and study its impact on protein release, we exposed six CRC cell lines to lipopolysaccharide (LPS) and investigated the effect on the secretome using in-depth mass spectrometry-based proteomics.

Experimental Design: Cancer cell secretome was harvested in bio-duplicate after LPS treatment, and separated in EV and soluble secretome (SS) fractions. Gel-fractionated proteins were analysed by label-free nano-liquid chromatography coupled to tandem mass spectrometry.

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Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles.

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Germline mutations in the Folliculin () tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. is a conserved, essential gene linked to diverse cellular processes but the mechanism by which prevents kidney cancer remains unknown. Here, we show that disrupting in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity.

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Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ≈50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, HCM), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, HCM). Genotype-specific differences have been reported in cardiac function.

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Article Synopsis
  • Pancreatic ductal adenocarcinoma (PDAC) is marked by a low number of cancer cells surrounded by a significant amount of stroma, which includes nontumor cells and other components and impacts patient outcomes.
  • A detailed proteomic analysis using laser-capture microdissection (LCM) revealed the largest set of proteins associated with PDAC, highlighting the importance of analyzing these distinct compartments separately.
  • The study identified specific proteins, such as CALB2 in tumor cells and COL11A1 in stroma, as potential prognostic markers, and examined the role of the tumor receptor EPHA2 in the behavior of cancer cells, demonstrating the necessity of compartment-specific analysis for understanding PDAC.
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Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML.

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Identification of predictive biomarkers for targeted therapies requires information on drug exposure at the target site as well as its effect on the signaling context of a tumor. To obtain more insight in the clinical mechanism of action of protein kinase inhibitors (PKIs), we studied tumor drug concentrations of protein kinase inhibitors (PKIs) and their effect on the tyrosine-(pTyr)-phosphoproteome in patients with advanced cancer. Tumor biopsies were obtained from 31 patients with advanced cancer before and after 2 weeks of treatment with sorafenib (SOR), erlotinib (ERL), dasatinib (DAS), vemurafenib (VEM), sunitinib (SUN) or everolimus (EVE).

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Article Synopsis
  • * In a study using mice, researchers examined how the presence or absence of PVAT affects muscle perfusion and glucose uptake during insulin stimulation, finding that PVAT is essential for proper IMVR.
  • * The removal of PVAT not only disrupted the blood flow to muscles but also altered important protein clusters involved in metabolism, indicating a significant role of PVAT in managing vascular health and metabolic processes relevant to conditions like obesity and diabetes.
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