The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL’s α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL’s catalytic site, including β2, β3–α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3–α3 and progress to β5 and β4–α4, ultimately leading to the irreversible unfolding of regions that form LPL’s catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL’s α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

House by the road: estate, dacha, railway in historical and literary aspects (19 – early 20 century)

The study highlights relationship between changes in material culture (development of railroad network), social infrastructure (spread of dacha villages) and poetics of literary works in Russia of the second half of the 19th – early 20th c., addressing “dacha topos”. The paper draws on the texts, which introduce railroad as a symbol of destruction of traditional values under the pressure of bourgeois “industrialism” and pernicious “infernalityˮ (А. М. Zhemchuzhnikov, F. M. Dostoevsky, L. N. Tolstoy, A. S. Serafimovich, А. А. Blok and others). The author shows that dacha, wrought by railroad civilization, is conceptualized as part of packed, petty-bourgeois, low-minded and soulless city as opposed to country estate as a lone “paradise on earth” and hermitage of high culture (А. P. Tchekhov, N. А. Leykin, А. P. Kamensky and others). The paper draws attention to metamorphoses of artistic time in passing from “estate topos” with inherent temporal static and cycliсity to “dacha topos” with precipitous and irreversible unfolding in time. The author concludes that the changes in artistic topics and temporality when addressing successive phenomena of estate and dacha are largely due to such new details of subjective figurativeness as the railroad and its attributes (locomotive, rails, wagons, anonymous passengers, travel speed etc.).

Influence of Calcium Ions on the Thermal Characteristics of α-amylase from Thermophilic Anoxybacillus sp. GXS-BL

Background: &#945;-Amylases are starch-degrading enzymes and used widely, the study on thermostability of &#945;-amylase is a central requirement for its application in life science and biotechnology. </P><P> Objective: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of &#945;-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. </P><P> Methods: &#945;-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. </P><P> Results: With calcium ions, the values of Topt, T50, t1/2, Tm and &#916;H in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on &#945;-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including &#945;- helical contents increasing, and the transition from &#945;-helices to &#946;-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. </P><P> Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

NMR studies reveal that protein dynamics critically mediate aggregation of the well-folded and very soluble E. coli S1 ribosomal protein

Unlike mammalian aging associated with many hallmarks, E. coli aging is only significantly characterized by protein aggregation, thus offering an excellent model for addressing the relationship between protein aggregation and aging. Here we characterized conformations, unfolding and dynamics of ribosomal protein S1 and its D3/D5 domains using NMR, CD and fluorescence spectroscopy. S1 is a 557-residue modular protein containing six S1 motifs. Paradoxically, while S1 is well-folded and very soluble in vitro, it was found in various lists of aggregated E. coli proteins. Our results decipher: 1) S1 has dynamic inter-domain interactions. Strikingly, S1 and its D3/D5 domains have significantly exposed hydrophobic patches characterized by irreversible unfolding. 2) Although D5 has significantly restricted backbone motion on ps-ns time scale, it has global μs-ms conformational dynamics and particularly high “global breathing” motions. 3) D5 assumes the conserved β-barrel fold but contains large hydrophobic patches at least dynamically accessible. Taken together, our study reveals that S1 could be prone to aggregation due to significant dynamics at two levels: inter-domain interactions and individual domains, which may even render buried hydrophobic patches/cores accessible for driving aggregation. This mechanism is most likely to operate in many proteins of E. coli and other organisms including human.