Denaturation via Surfactants Changes Composition of Protein Corona.

The use of nanocarriers as drug delivery vehicles brings them into contact with blood plasma proteins. Polymeric nanocarriers require some sort of surfactant to ensure colloidal stability. Formation of the protein corona is therefore determined not only by the intrinsic properties of the nanocarrier itself but also by the accompanying surfactant. Although it is well-known that surfactants have an impact on protein structure, only few studies were conducted on the specific effect of surfactants on the composition of protein corona of nanocarriers. Therefore, we analyzed the composition of the protein corona on "stealth" nanoparticles with additional surfactant (cetyltrimethylammonium chloride, CTMA-Cl) after plasma incubation. Additional CTMA-Cl led to an enrichment of apolipoprotein-A1 and vitronectin in the corona, while less clusterin could be found. Further, the structural stability of apolipoprotein-A1 and clusterin was monitored for a wide range of CTMA-Cl concentrations. Clusterin turned out to be more sensitive to CTMA-Cl, with denaturation occurring at lower concentrations.

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

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 ß-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.

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.

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°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.

Exposure of vital cells to necrotic cell lysates induce the IRE1α branch of the unfolded protein response and cell proliferation.

Necrosis is a form of cell death that is detrimental to the affected tissue because the cell ruptures and releases its content (reactive oxygen species among others) into the extracellular space. Clusterin (CLU), a cytoprotective extracellular chaperone has been shown to be upregulated in the face of necrosis. We here show that in addition to CLU upregulation, necrotic cell lysates induce JNK/SAPK signaling, the IRE1α branch of the unfolded protein response (UPR), the MAPK/ERK1/2, and the mTOR signaling pathways and results in an enhanced proliferation of the vital surrounding cells. We name this novel response mechanism: Necrosis-induced Proliferation (NiP).

The CLU-files: disentanglement of a mystery.

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.

Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-κB signaling pathway.

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-κB-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 in vitro 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, CLU1­449, CLU21­449 and CLU34­449, which all reside in the cytosol of unstressed and stressed HEK­293 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-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.