19 research outputs found

    Assessment of the role of clusterin/apolipoprotein J in Cellular Homeostasis

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    Clusterin (CLU) ist ein stark glykosyliertes extrazellulĂ€res Chaperone welches beinahe ubiquitĂ€r in Vertebraten und Geweben exprimiert wird. Seit seiner Entdeckung vor mehr als 30 Jahren wurde insbesondere die sezernierte Form von CLU (sCLU) als zytoprotektives Protein in starker Korrelation mit Erkrankungen wie Krebs, Arteriosklerose und der Alzheimer Erkrankung betrachtet. Trotz intensiver Erforschung (gegenwĂ€rtig rund 2500 Artikel in der PubMed) ist seine exakte Rolle in Erkrankungen nach wie vor unbekannt. Die vorliegende Arbeit fokussiert sich primĂ€r auf die Rolle von sCLU in der zellulĂ€ren Homöostase und Proteostase. Grundlegende besondere Eigenschaften menschlichen sCLU‘s sind dessen Glykosylierung und seine proteolytische Spaltung in zwei separate Ketten, welche durch DisulfidbrĂŒcken verbunden bleiben. ZunĂ€chst wird daher die Rolle dieser strukturellen Eigenschaften fĂŒr die Faltung und ChaperonaktivitĂ€t von sCLU beleuchtet. Die experimentelle DurchfĂŒhrung dessen erfolgte primĂ€r mit menschlichen und murinen sCLU. In diesem Zusammenhang wird weiterhin bewertet auf welche Weise CLU im Tierreich konserviert ist und ob CLU tatsĂ€chlich als konserviertes sezerniertes molekulares Chaperon betrachtet werden kann. Basierend auf Erkenntnissen ĂŒber die ChaperonaktivitĂ€t von sCLU wird eine potentielle Rolle in der Zellbiologie diskutiert. Es muss dabei zur Kenntnis genommen werden, dass vorangegangene Studien eine erhöhte Menge von CLU und sCLU beim Auftreten von Pathologien mit unkontrollierten sowie schĂ€digenden Zelltodereignissen wie bei IschĂ€mien, EntzĂŒndungen oder Verletzungen beobachteten. Der zweite Abschnitt wird sich daher mit der Expression von CLU in einem In vitro-Nekrosemodell befassen um seine Funktion unter diesen Bedingungen zu klĂ€ren. Interessanterweise fĂŒhrte dieser Ansatz zu neuen Einblicken ĂŒber CLU‘s Rolle in der „Antwort auf ungefaltete Proteine“ (englisch: Unfolded Protein Response, UPR) und offenbarte CLU‘s Zusammenspiel mit verschiedenen proteostatischen Pfaden. ZusĂ€tzlich wird ein neuer zellulĂ€rer Mechanismus namens Nekrose-induzierte Proliferation (englisch: Necrosis-induced Proliferation, NiP) vorgeschlagen, welcher bedeutende Konsequenzen in der Behandlung von Krebs haben könnte. Zusammenfassend diskutiert dieser Aufsatz grundlegende Aspekte der Biochemie und Zellbiologie von CLU um dessen Rolle in Krankheit und Tod besser zu verstehen.Clusterin (CLU) is a highly glycosylated extracellular chaperone with a nearly ubiquitous expression in vertebrates and tissues. Since its discovery more than three decades ago, especially the secretory form of CLU (sCLU) was considered being a cytoprotective protein with high correlation to certain diseases, such as cancer, atherosclerosis and Alzheimer’s disease. Despite of intense research activity (almost 2500 articles in PubMed), its exact role in diseases is still unknown. This thesis focuses primarily on the role of sCLU in the cellular homeostasis and proteostasis. Underlying important properties of human sCLU are the glycosylation and the proteolytic cleavage into two separate chains held together by disulfide bonds. Therefore, at first the role of these structural properties on the folding and chaperone activity of sCLU will be addressed. This will be predominately conducted by using human and mouse sCLU. In this context, it will be further assessed in which way CLU is conserved among the animal kingdom and whether the CLU protein indeed can be considered as a conserved secretory molecular chaperone. Based on findings concerning the chaperone activity of sCLU, a potential role in cell biology will be addressed. Noticeable, in past studies an elevated abundance of CLU and sCLU was identified by virtue of its occurrence in pathologies with uncontrolled or detrimental cell death, such as in ischemia, inflammation or wounding. The second section therefore investigates the expression of CLU in an in vitro necrosis model to understand its function in harmful cellular settings. Intriguingly, this approach led to new insights concerning CLU’s role in the unfolded protein response (UPR) and revealed CLUs’ interplay with various proteostatic pathways. Moreover, a new cellular mechanism called Necrosis-induced Proliferation (NiP) will be proposed, which might have important implications for treatment of cancer. In summary, this thesis discusses basic aspects of CLU biochemical and cell biological properties to understand its role in death and disease

    The CLU-files: disentanglement of a mystery.

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    The multifaceted protein clusterin (CLU) has been challenging researchers for more than 35 years. The characterization of CLU as a molecular chaperone was one of the major breakthroughs in CLU research. Today, secretory clusterin (sCLU), also known as apolipoprotein J (apoJ), is considered one of the most important extracellular chaperones ever found. It is involved in a broad range of physiological and pathophysiological functions, where it exerts a cytoprotective role. Descriptions of various forms of intracellular CLU have led to further and even contradictory functions. To untangle the current state of knowledge of CLU, this review will combine old views in the field, with new discoveries to highlight the nature and function of this fascinating protein(s). In this review, we further describe the expression and subcellular location of various CLU forms. Moreover, we discuss recent insights into the structure of CLU and assess how structural properties as well as the redox environment determine the chaperone activity of CLU. Eventually, the review connects the biochemistry and molecular cell biology of CLU with medical aspects, to formulate a hypothesis of a CLU function in health and disease

    The chaperone activity of clusterin is dependent on glycosylation and redox environment

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    Background/Aims: Clusterin (CLU), also known as Apolipoprotein J (ApoJ) is a highly glycosylated extracellular chaperone. In humans it is expressed from a broad spectrum of tissues and related to a plethora of physiological and pathophysiological processes, such as Alzheimer's disease, atherosclerosis and cancer. In its dominant form it is expressed as a secretory protein (secreted CLU, sCLU). During its maturation, the sCLU-precursor is N-glycosylated and cleaved into an α- and a ÎČ-chain, which are connected by five symmetrical disulfide bonds. Recently, it has been demonstrated that besides the predominant sCLU, rare intracellular CLU forms are expressed in stressed cells. Since these forms do not enter or complete the secretory pathway, they are not proteolytically modified and show either no or only core glycosylation. Due to their sparsity, these intracellular forms are functionally poorly characterized. To evaluate the function(s) of these stress-related intracellular forms, we investigate for the first time the impact of proteolytic cleavage, differential glycosylation and the influence of the redox environment on the chaperone activity of CLU. Methods: Non-cleavable sCLU was generated by expression from a mutant construct of sCLU, in which the furin-like proprotein convertase (PC) recognition site was modified. After purification of recombinant uncleaved sCLU from the medium of over-expressing cells, we performed chaperone activity assays to compare the activities of wild-type (cleaved) and uncleaved mutant sCLU. Additionally, this approach enabled us to investigate the role of carbohydrates, the proteolytic maturation and reducing conditions on CLU chaperone activity. Further, we characterized the differentially treated CLU forms by using MALDI-TOF, CD-spectroscopy and Western blotting in addition to the functional assay. Results: We show that the PC-cleavage is dispensable for sCLU chaperone activity. Moreover, our data demonstrate that while fully deglycosylated sCLU lacks chaperone activity, partially deglycosylated sCLU is still capable of solubilizing target proteins. Most importantly, we here demonstrate for the first time that uncleaved sCLU is highly sensitive towards reducing conditions. Conclusions: Our study provides evidence that unglycosylated intracellular CLU forms cannot exhibit a chaperone activity compared to sCLU. Additionally, we support recent postulates that glycosylated intracellular CLU forms may act as a redox sensor under oxidative stress conditions. Furthermore, we conclude that the proteolytic cleavage of sCLU is important to maintain full chaperone activity, i.e. in the presence of necrosis

    Impact of green and blue‐green light on the growth, pigment concentration, and fatty acid unsaturation in the microalga Monoraphidium braunii

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    The spectral composition of light is an important factor for the metabolism of photosynthetic organisms. Several blue light-regulated metabolic processes have already been identified in the industrially relevant microalga Monoraphidium braunii. However, little is known about the spectral impact on this species' growth, fatty acid (FA), and pigment composition. In this study, M. braunii was cultivated under different light spectra (white light: 400–700 nm, blue light: 400–550 nm, green light: 450–600 nm, and red light: 580–700 nm) at 25°C for 96 h. The growth was monitored daily. Additionally, the FA composition, and pigment concentration was analyzed after 96 h. The highest biomass production was observed upon white light and red light irradiation. However, green light also led to comparably high biomass production, fueling the scientific debate about the contribution of weakly absorbed light wavelengths to microalgal biomass production. All light spectra (white, blue, and green) that comprised blue-green light (450–550 nm) led to a higher degree of FA unsaturation and a greater concentration of all identified pigments than red light. These results further contribute to the growing understanding that blue-green light is an essential trigger for maximized pigment concentration and FA unsaturation in green microalgae

    Blue‐green light is required for a maximized fatty acid unsaturation and pigment concentration in the microalga Acutodesmus obliquus

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    Blue-green light is known to maximize the degree of fatty acid (FA) unsaturation in microalgae. However, knowledge on the particular waveband responsible for this stimulation of FA desaturation and its impact on the pigment composition in microalgae remains limited. In this study, Acutodesmus obliquus was cultivated for 96 h at 15 degrees C with different light spectra (380-700 nm, 470-700 nm, 520-700 nm, 600-700 nm, and dark controls). Growth was monitored daily, and qualitative characterization of the microalgal FA composition was achieved via gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS). Additionally, a quantitative analysis of microalgal pigments was performed using high-performance liquid chromatography with diode array detection (HPLC-DAD). Spectra that included wavelengths between 470 and 520 nm led to a significantly higher percentage of the polyunsaturated fatty acids (PUFA) 18:3 and 16:4, compared to all other light conditions. However, no significant differences between the red light cultivations and the heterotrophic dark controls were observed for the FA 18:3 and 16:4. These results indicate, that exclusively the blue-green light waveband between 470 and 520 nm is responsible for a maximized FA unsaturation in A. obliquus. Furthermore, the growth and production of pigments were impaired if blue-green light (380-520 nm) was absent in the light spectrum. This knowledge can contribute to achieving a suitable microalgal pigment and FA composition for industrial purposes and must be considered in spectrally selective microalgae cultivation systems

    Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-ÎșB Signaling Pathway

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    <div><p>Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-ÎșB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with <i>in vitro</i> mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU<sub>1‑449</sub>, CLU<sub>21‑449</sub> and CLU<sub>34‑449</sub>, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-ÎșB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.</p> </div

    Proteasomal inhibition and heat stress modulate sCLU and intracellular CLU protein expression in cancer and non-cancer cells.

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    <p>HEK‑293, PC‑3, MCF‑7 and Caco-2 cells were treated with DMSO as control (C), 10 ”M MG-132 (MG) or subjected to heat shock (45°C). Whole cell lysates (upper panel) and cell culture media (lower panel) of cells were analyzed for CLU expression by Western blot. 45-50 kDa CLU protein bands were detected primarily in stressed cells (*). Data shown are representative of three independent experiments.</p
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