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Träfflista för sökning "WFRF:(Lagerstedt Jens O.) ;pers:(Del Giudice Rita)"

Sökning: WFRF:(Lagerstedt Jens O.) > Del Giudice Rita

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1.
  • Del Giudice, Rita, et al. (författare)
  • High-efficient bacterial production of human ApoA-I amyloidogenic variants
  • 2018
  • Ingår i: Protein Science. - : Wiley. - 0961-8368. ; 27:12, s. 2101-2109
  • Tidskriftsartikel (refereegranskat)abstract
    • Apolipoprotein A-I (ApoA-I)-related amyloidosis is a rare disease caused by missense mutations in the APOA1 gene. These mutations lead to protein aggregation and abnormal accumulation of ApoA-I amyloid fibrils in heart, liver, kidneys, skin, nerves, ovaries, or testes. Consequently, the carriers are at risk of single- or multi-organ failure and of need of organ transplantation. Understanding the basic molecular structure and function of ApoA-I amyloidogenic variants, as well as their biological effects, is, therefore, of great interest. However, the intrinsic low stability of this type of proteins makes their overexpression and purification difficult. To overcome this barrier, we here describe an optimized production and purification procedure for human ApoA-I amyloidogenic proteins that efficiently provides between 46 mg and 91 mg (depending on the protein variant) of pure protein per liter of Escherichia coli culture. Structural integrity of the amyloidogenic and native ApoA-I proteins were verified by circular dichroism spectroscopy and intrinsic fluorescence analysis, and preserved functionality was demonstrated by use of a lipid clearance assay as well as by reconstitution of high-density lipoprotein (HDL) particles. In conclusion, the use of the described high-yield protein production system to obtain amyloidogenic ApoA-I proteins, and their native counterpart, will enable molecular and cellular experimental studies aimed to explain the molecular basis for this rare disease.
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2.
  • Del Giudice, Rita, et al. (författare)
  • Structural determinants in ApoA-I amyloidogenic variants explain improved cholesterol metabolism despite low HDL levels
  • 2017
  • Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002. ; 1863:12, s. 3038-3048
  • Tidskriftsartikel (refereegranskat)abstract
    • Twenty Apolipoprotein A-I (ApoA-I) variants are responsible for a systemic hereditary amyloidosis in which protein fibrils can accumulate in different organs, leading to their failure. Several ApoA-I amyloidogenic mutations are also associated with hypoalphalipoproteinemia, low ApoA-I and high-density lipoprotein (HDL)-cholesterol plasma levels; however, subjects affected by ApoA-I-related amyloidosis do not show a higher risk of cardiovascular diseases (CVD). The structural features, the lipid binding properties and the functionality of four ApoA-I amyloidogenic variants were therefore inspected in order to clarify the paradox observed in the clinical phenotype of the affected subjects. Our results show that ApoA-I amyloidogenic variants are characterized by a different oligomerization pattern and that the position of the mutation in the ApoA-I sequence affects the molecular structure of the formed HDL particles. Although lipidation increases ApoA-I proteins stability, all the amyloidogenic variants analyzed show a lower affinity for lipids, both in vitro and in ex vivo mouse serum. Interestingly, the lower efficiency at forming HDL particles is compensated by a higher efficiency at catalysing cholesterol efflux from macrophages. The decreased affinity of ApoA-I amyloidogenic variants for lipids, together with the increased efficiency in the cholesterol efflux process, could explain why, despite the unfavourable lipid profile, patients affected by ApoA-I related amyloidosis do not show a higher CVD risk.
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3.
  • Del Giudice, Rita, et al. (författare)
  • The Apparent Organ-Specificity of Amyloidogenic ApoA-I Variants Is Linked to Tissue-Specific Extracellular Matrix Components
  • 2023
  • Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Apolipoprotein A-I (ApoA-I) amyloidosis is a rare protein misfolding disease where fibrils of the N-terminal domain of the protein accumulate in several organs, leading to their failure. Although ApoA-I amyloidosis is systemic, the different amyloidogenic variants show a preferential tissue accumulation that appears to correlate with the location of the mutation in the protein sequence and with the local extracellular microenvironment. However, the factors leading to cell/tissues damage, as well as the mechanisms behind the observed organ specificity are mostly unknown. Therefore, we investigated the impact of ApoA-I variants on cell physiology and the mechanisms driving the observed tissue specificity. We focused on four ApoA-I amyloidogenic variants and analyzed their cytotoxicity as well as their ability to alter redox homeostasis in cell lines from different tissues (liver, kidney, heart, skin). Moreover, variant-specific interactions with extracellular matrix (ECM) components were measured by synchrotron radiation circular dichroism and enzyme-linked immunosorbent assay. Data indicated that ApoA-I variants exerted a cytotoxic effect in a time and cell-type-specific manner that seems to be due to protein accumulation in lysosomes. Interestingly, the ApoA-I variants exhibited specific preferential binding to the ECM components, reflecting their tissue accumulation pattern in vivo. While the binding did not to appear to affect protein conformations in solution, extended incubation of the amyloidogenic variants in the presence of different ECM components resulted in different aggregation propensity and aggregation patterns.
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4.
  • Domingo-Espín, Joan, et al. (författare)
  • Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism
  • 2018
  • Ingår i: Biochimica et Biophysica Acta - Molecular Basis of Disease. - : Elsevier BV. - 0925-4439. ; 1864:9, s. 2822-2834
  • Tidskriftsartikel (refereegranskat)abstract
    • Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated. ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion. Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.
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5.
  • Giudice, Rita Del, et al. (författare)
  • Synchrotron radiation circular dichroism spectroscopy reveals structural divergences in HDL-bound apoA-I variants
  • 2017
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) provides cardiovascular protection. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to analyze the dynamic solution structure of the apoA-I protein in the apo- and HDL-states and the protein structure conversion in HDL formation. Wild-type apoA-I protein was compared to human variants that either are protective (R173C, Milano) or lead to increased risk for ischaemic heart disease (A164S). Comparable secondary structure distributions in the HDL particles, including significant levels of beta strand/turn, were observed. ApoA-I Milano in HDL displayed larger size heterogeneity, increased protein flexibility, and an altered lipid-binding profile, whereas the apoA-I A164S in HDL showed decrease thermal stability, potentially linking the intrinsic HDL propensities of the variants to disease risk.
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6.
  • Miskelly, Michael G., et al. (författare)
  • RNA sequencing unravels novel L cell constituents and mechanisms of GLP-1 secretion in human gastric bypass-operated intestine
  • 2024
  • Ingår i: Diabetologia. - : Springer. - 0012-186X .- 1432-0428. ; 67:2, s. 356-370
  • Tidskriftsartikel (refereegranskat)abstract
    • Aims/hypothesis: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo.Methods: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1-/- mice and immunohistochemistry and gene expression analyses were performed ex vivo.Results: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca2+ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1-/-, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression.Conclusions/interpretation: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1.
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7.
  • Nilsson, Oktawia, et al. (författare)
  • Apolipoprotein A-I primes beta cells to increase glucose stimulated insulin secretion
  • 2020
  • Ingår i: Biochimica et Biophysica Acta - Molecular Basis of Disease. - : Elsevier BV. - 0925-4439. ; 1866:3
  • Tidskriftsartikel (refereegranskat)abstract
    • The increase of plasma levels of high-density lipoproteins and Apolipoprotein A-I (ApoA-I), its main protein component, has been shown to have a positive action on glucose disposal in type 2 diabetic patients. The current study investigates the unexplored function of ApoA-I to prime beta cells for improved insulin secretion. INS-1E rat clonal beta cells as well as isolated murine islets were used to study the effect of ApoA-I on responsiveness of the beta cells to high glucose challenge. Confocal and transmission electron microscopy were used to dissect ApoA-I mechanisms of action. Chemical endocytosis blockers were used to understand the role of ApoA-I internalization in mediating its positive effect. Pre-incubation of beta cells and isolated murine islets with ApoA-I augmented glucose stimulated insulin secretion. This effect appeared to be due to an increased reservoir of insulin granules at the cell membrane, as confirmed by confocal and transmission electron microscopy. Moreover, ApoA-I induced pancreatic and duodenal homeobox 1 (PDX1) shuttling from the cytoplasm to the nucleus, with the subsequent increase in the proinsulin processing enzyme protein convertase 1 (PC1/3). Finally, the blockade of ApoA-I endocytosis in beta cells resulted in a loss of ApoA-I positive action on insulin secretion. The proposed mechanisms of the phenomenon here described include ApoA-I internalization into beta cells, PDX1 nuclear translocation, and increased levels of proinsulin processing enzymes. Altogether, these events lead to an increased number of insulin granules.
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8.
  • Nilsson, Oktawia, et al. (författare)
  • Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux
  • 2021
  • Ingår i: Journal of Lipid Research. - 0022-2275. ; 62
  • Tidskriftsartikel (refereegranskat)abstract
    • Apolipoprotein A-I (ApoA-I) of high density lipoproteins (HDLs) is essential for the transportation of cholesterol between peripheral tissues and the liver. However, specific mutations in ApoA-I of HDLs are responsible for a late-onset systemic amyloidosis, the pathological accumulation of protein fibrils in tissues and organs. Carriers of these mutations do not exhibit increased cardiovascular disease risk despite displaying reduced levels of ApoA-I/ HDL cholesterol. To explain this paradox, we show that the HDL particle profiles of patients carrying either L75P or L174S ApoA-I amyloidogenic variants show a higher relative abundance of the 8.4-nm versus 9.6-nm particles and that serum from patients, as well as reconstituted 8.4- and 9.6-nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogendeuterium exchange revealed that the variants in 8.4-nm rHDL have altered secondary structure composition and display a more flexible binding to lipids than their native counterpart. The reduced HDL cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles, and better cholesterol efflux due to altered, region-specific protein structure dynamics.
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9.
  • Nilsson, Oktawia, et al. (författare)
  • Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux
  • 2021
  • Ingår i: Journal of Lipid Research. - : Elsevier. - 0022-2275 .- 1539-7262. ; 62
  • Tidskriftsartikel (refereegranskat)abstract
    • Apolipoprotein A-I (ApoA-I) of high density lipoproteins (HDLs) is essential for the transportation of cholesterol between peripheral tissues and the liver. However, specific mutations in ApoA-I of HDLs are responsible for a late-onset systemic amyloidosis, the pathological accumulation of protein fibrils in tissues and organs. Carriers of these mutations do not exhibit increased cardiovascular disease risk despite displaying reduced levels of ApoA-I/HDL cholesterol. To explain this paradox, we show that the HDL particle profiles of patients carrying either L75P or L174S ApoA-I amyloidogenic variants show a higher relative abundance of the 8.4-nm versus 9.6-nm particles and that serum from patients, as well as reconstituted 8.4- and 9.6-nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4-nm rHDL have altered secondary structure composition and display a more flexible binding to lipids than their native counterpart. The reduced HDL cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles, and better cholesterol efflux due to altered, region-specific protein structure dynamics.
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10.
  • Svensson, Daniel, et al. (författare)
  • Apolipoprotein A-I attenuates LL-37-induced endothelial cell cytotoxicity
  • 2017
  • Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 493:1, s. 71-76
  • Tidskriftsartikel (refereegranskat)abstract
    • The human cathelicidin peptide LL-37 has antimicrobial and anti-biofilm functions, but LL-37 may also damage the host by triggering inflammation and exerting a cytotoxic effect, thereby reducing host cell viability. Human plasma mitigates LL-37-induced host cell cytotoxicity but the underlying mechanisms are not completely understood. Apolipoprotein A-I (ApoA-I) is a plasma protein endowed with atheroprotective effects. Here, we investigate the interaction between ApoA-I and LL-37 by biochemical techniques, and furthermore assess if ApoA-I protects against LL-37-evoked cytotoxicity in human umbilical vein endothelial cells (HUVEC). Our results demonstrated that ApoA-I effectively binds LL-37. The binding of ApoA-I to LL-37 resulted in a structural rearrangement of the protein, but this interaction did not cause lower ApoA-I stability. Recombinant ApoA-I protected against LL-37-induced cytotoxicity in HUVEC and endogenous ApoA-I knockdown in HepG2 cells made the cells more sensitive to LL-37-evoked cytotoxicity. We conclude that ApoA-I physically interacts with LL-37 and antagonizes LL-37-induced down-regulation of endothelial cell viability suggesting that this mechanism counteracts endothelial cell dysfunction.
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