Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a ‘molecular tweezer’ specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion–amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.DOI: http://dx.doi.org/10.7554/eLife.05397.001
The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis Nature Chemical Biology, 15 (7): 710-720 Editorial SummaryThe cholesterol transfer protein GRAMD1A was identified as the target of the autophagy inhibitors autogramin-1 and 2. GRAMD1A is required for autophagosome biogenesis, and autogramins represent tool compounds for studying this process. AbstractAutophagy mediates the degradation of damaged proteins, organelles and pathogens and plays a key role in health and disease. The identification of new mechanisms involved in autophagy regulation is of major interest. In particular little is known about the roles of lipids and lipid binding proteins in the early steps of autophagosome biogenesis. Through target agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a novel autophagy inhibitor class. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain containing protein 1A (GRAMD1A), which had not been implicated in autophagy before, and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a novel biological function of GRAMD1A and a new role for cholesterol in autophagy.
Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the “phosphate tweezer” CLR01, a “carboxylate tweezer” CLR05, and a “phosphate clip” PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.
We studied the combined effects of pressure (0.1-200 MPa) and temperature (22, 30, and 38 8 8C) on the catalytic activity of designed amyloid fibrils using ah igh-pressure stopped-flows ystem with rapid UV/Vis absorption detection. Complementary FT-IR spectroscopic data revealed ar emarkably high pressure and temperature stability of the fibrillar systems.H igh pressure enhances the esterase activity as ac onsequence of an egative activation volume at all temperatures (about À14 cm 3 mol À1 ). The enhancement is sustained in the whole temperature range covered, which allows af urther acceleration of the enzymatic activity at high temperatures (activation energy 45-60 kJ mol À1 ). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils.Amyloid fibrils are linear polypeptide aggregates with ah ighly periodic cross-b sheet conformation, which abnormally occur inside the body and are associated with diseases such as Alzheimersa nd Parkinsons. [1] Increasing evidence shows,h owever,t hat amyloid structures may also occur as functional biological units in vivo,a nd there is considerable interest in the material sciences and bionanotechnology to exploit the unique structural features of these highly stable, nanostructured, and biodegradable assemblies for the generation of novel applications and technical tools,s uch as protein-based catalysis,conducting nanowires,and devices for water purification. [2] Ak ey factor limiting the further utility of amyloid materials and their incorporation into technical devices is their so far unclear stability and performance under processrelevant conditions.T oe stablish these characteristics we herein describe the results obtained by exposing two recently described preparations of de novo designed and catalytically active fibril systems to different types of environmental stress. These two systems consist of the peptides Ac-LHLHLRL-CONH 2 (AF1) and Ac-IHIHIQI-CONH 2 (AF2) that form amyloid fibrils in vitro,w hich exhibit, upon Zn 2+ binding,a n esterase activity that can be spectroscopically followed by the hydrolysis of the substrate p-nitrophenyl acetate (pNPA). [3] Tr ansmission electron microscopy (TEM) showed that the fibril morphology of both samples is markedly different. Both samples contain polymorphic fibrils,a sc an be inferred from the broad variations of the fibril width (Figure 1), and there are considerable variations in the quaternary structure of the fibrils in these samples.A F1 fibrils are relatively long and twisted ribbons ( Figure 1A), while AF2 fibrils exhibit amore plate-like morphology ( Figure 1B).We then determined the effect of different environmental conditions,s uch as high hydrostatic pressure (HHP) and temperature,o nt he structural stability and catalytic activity of the two fibril enzymes.U ntil now the effects of HHP on enzymatic substrate conversions have been characterized only sporadically. [4] HHP would be expected to accelerate enzymatic reactions,i f...
Owing to the presence of various types of osmolytes in the cellular environment, this study focuses on the impact of stabilizing (TMAO and betaine) as well as destabilizing (urea) cosolvents on the aggregation and fibrillation reaction of the highly amyloidogenic islet amyloid polypeptide (IAPP). IAPP is associated with type-2 diabetes mellitus and is responsible for the disease accompanying β-cell membrane permeabilization and final β-cell loss. To reveal the impact of the cosolvents on the aggregation kinetics, conformational and morphological changes upon IAPP fibrillation, Thioflavin T fluorescence spectroscopy, atomic force microscopy and attenuated total reflection Fourier-transform infrared spectroscopy were applied. For TMAO, and less pronounced for betaine, a decrease of the growth rate of fibrils is observed, whereas the lag phase remains essentially unchanged, indicating the ability of the compatible solutes to stabilize large oligomeric and protofibrillar structures and therefore hamper fibril elongation. Conversely, urea displays concentration-dependent prolongation of the lag phase, indicating stabilization of IAPP in its unfolded monomeric state, hence leading to retardation of IAPP nuclei formation. Mixtures of urea with TMAO, and to a lesser extent with betaine, exhibit a counteractive effect. TMAO is able to fully compensate the prolonged lag phase induced by urea. This strongly matches the findings of a counteraction of TMAO and urea in protein folding and unfolding experiments. The data also reveal that the influence of these cosolvents is only on the aggregation kinetics without markedly changing the final IAPP fibrillar morphology, i.e., the solution structure and cosolvent composition essentially affect the kinetics of the fibrillation process only.
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