Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

From a Droplet to a Fibril and from a Fibril to a Droplet: Intertwined Transition Pathways in Highly Dynamic Enzyme-Modulated Peptide-Adenosine Triphosphate Systems.

Many cellular coassemblies of proteins and polynucleotides facilitate liquid-liquid phase separation (LLPS) and the subsequent self-assembly of disease-associated amyloid fibrils within the liquid droplets. Here, we explore the dynamics of coupled phase and conformational transitions of model adenosine triphosphate (ATP)-binding peptides, ACC1-13Kn, consisting of the potent amyloidogenic fragment of insulin's A-chain (ACC1-13) merged with oligolysine segments of various lengths (Kn, n = 16, 24, 40). The self-assembly of ATP-stabilized amyloid fibrils is preceded by LLPS for peptides with sufficiently long oligolysine segments. The two-component droplets and fibrils are in dynamic equilibria with free ATP and monomeric peptides, which makes them susceptible to ATP-hydrolyzing apyrase and ACC1-13Kn-digesting proteinase K. Both enzymes are capable of rapid disassembly of amyloid fibrils, producing either monomers of the peptide (apyrase) or free ATP released together with cleaved-off oligolysine segments (proteinase K). In the latter case, the enzyme-sequestered Kn segments form subsequent droplets with the co-released ATP, resulting in an unusual fibril-to-droplet transition. In support of the highly dynamic nature of the aggregate-monomer equilibria, addition of superstoichiometric amounts of free peptide to the ACC1-13Kn-ATP coaggregate causes its disassembly. Our results show that the droplet state is not merely an intermediate phase on the pathway to the amyloid aggregate but may also constitute the final phase of a complex amyloidogenic protein misfolding scenario rich in highly degraded protein fragments incompetent to transition again into fibrils.

Alcohol-Induced Conformation Changes and Thermodynamic Signatures in the Binding of Polyphenols to Proline-Rich Salivary Proteins.

The first contact of polyphenols (tannins) with the human body occurs in the mouth, where they are known to interact with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for the development of astringency, but affect also various other biochemical processes. Employing thermodynamic measurements, fluorescence and CD spectroscopy, we investigated the binding process of the prototypical polyphenol ellagic acid (EA) to different IB-PRPs and BSA, also in the presence of ethanol, which is known to influence tannin-protein interactions. Binding of EA to BSA and the small peptide IB7-14 is weak, but very strong to IB9-37. The differences in binding strength and stoichiometry are due to differences in the binding motifs, which also lead to differences in the thermodynamic signatures of the binding process. EA binding to BSA is enthalpy-driven, whereas binding to both IB7-14 and IB9-37 is entropy-driven. The presence of 10 vol.% EtOH, as present in wines, increases the binding constant of EA with BSA and IB7-14 drastically, but not that with IB9-37; however, it changes the binding stoichiometry. These differences can be attributed to the effect of EtOH on the conformation dynamics of the proteins and to changes in hydration properties in alcoholic solution.

The anticancer peptide LL-III binds with nanomolar affinity to human telomeric and cMyc G-quadruplexes.

LL-III is a natural anticancer peptide able to cross the membrane of cancer cells and to localize in the nucleolus, but its intracellular target is unknown. Here, we show that LL-III is able to bind with nM affinity to specific G-quadruplex structures known to be relevant anticancer targets.

Deep sea osmolytes in action: their effect on protein-ligand binding under high pressure stress.

Because organisms living in the deep sea and in the sub-seafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding reactions, must be adjusted to keep the associated volume changes low in order to function efficiently. Almost all organisms use organic cosolvents (osmolytes) to protect their cells from adverse environmental conditions. They counteract osmotic imbalance, stabilize the structure of proteins and maintain their function. We studied the binding properties of the prototypical ligand proflavine to two serum proteins with different binding pockets, BSA and HSA, in the presence of two prominent osmolytes, trimethylamine-N-oxide (TMAO) and glycine betaine (GB). TMAO and GB play an important role in the regulation and adaptation of life in deep-sea organisms. To this end, pressure dependent fluorescence spectroscopy was applied, supplemented by circular dichroism (CD) spectroscopy and computer modeling studies. The pressure-dependent measurements were also performed to investigate the intimate nature of the complex formation in relation to hydration and packing changes caused by the presence of the osmolytes. We show that TMAO and GB are able to modulate the ligand binding process in specific ways. Depending on the chemical make-up of the protein's binding pocket and thus the thermodynamic forces driving the binding process, there are osmolytes with specific interaction sites and binding strengths with water that are able to mediate efficient ligand binding even under external stress conditions. In the binding of proflavine to BSA and HSA, the addition of both compatible osmolytes leads to an increase in the binding constant upon pressurization, with TMAO being the most efficient, rendering the binding process also insensitive to pressurization even up to 2 kbar as the volume change remains close to zero. This effect can be corroborated by the effects the cosolvents impose on the strength and dynamics of hydration water as well as on the conformational dynamics of the protein.

Modulation of the Conformational Space of SARS-CoV-2 RNA Quadruplex RG-1 by Cellular Components and the Amyloidogenic Peptides α-Synuclein and hIAPP.

Given the emergence of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which particularly threatens older people with comorbidities such as diabetes mellitus and dementia, understanding the relationship between Covid-19 and other diseases is an important factor for treatment. Possible targets for medical intervention include G-quadruplexes (G4Qs) and their protein interaction partners. We investigated the stability and conformational space of the RG-1 RNA-G-quadruplex of the SARS-CoV-2 N-gene in the presence of salts, cosolutes, crowders and intrinsically disordered peptides, focusing on α-Synuclein and the human islet amyloid polypeptide, which are involved in Parkinson's disease (PD) and type-II diabetes mellitus (T2DM), respectively. We found that the conformational dynamics of the RG-1 G4Q is strongly affected by the various solution conditions. Further, the amyloidogenic peptides were found to strongly modulate the conformational equilibrium of the RG-1. Considerable changes are observed with respect to their interaction with human telomeric G4Qs, which adopt different topologies. These results may therefore shed more light on the relationship between PD as well as T2DM and the SARS-CoV-2 disease and their molecular underpinnings. Since dysregulation of G4Q formation by rationally designed targeting compounds affects the control of cellular processes, this study should contribute to the development of specific ligands for intervention.

Bipolar Imidazolium-Based Lipid Analogues for Artificial Archaeosomes.

Archaeal lipids have harvested biomedical and biotechnological interest because of their ability to form membranes with low permeability and enhanced temperature and pressure stability. Because of problems in isolating archaeal lipids, chemical synthesis appears to be a suitable means of producing model lipids that mimic the biological counterparts. Here, we introduce a new concept: we synthesized bipolar alkylated imidazolium salts of different chain lengths (BIm10-32) and studied their structure and lyotropic phase behavior. Furthermore, mixtures of the bolalipid analogues with phospholipid model biomembranes of diverse complexity were studied. DSC, fluorescence and FTIR spectroscopy, confocal fluorescence microscopy, DLS, SAXS, and TEM were used to reveal changes in lipid phase behavior, fluidity, the lipid's conformational order, and membrane morphology over a wide range of temperatures and for selected pressures. It could be shown that the long-chain BImN32 can form monolayer sheets. Integrated in phospholipid membranes, it reveals a fluidizing effect. Here, the two polar head groups, connected by a long alkyl chain, enable the integration into the bilayer. Interestingly, addition of BImN32 to fluid DPPC liposomes increased the lipid packing markedly, rendering the membrane system more stable at higher temperatures. The membrane system is also stable against compression as indicated by the high-pressure stability of the system, mimicking an archaeal lipid-like behavior. BImN32 incorporation into raft-like anionic model biomembranes led to marked changes in lateral membrane organization, topology, and fusogenicity of the membrane. Overall, it was found that long-chain imidazolium-based bolalipid analogues can help adjust membrane's biophysical properties, while the imidazolium headgroup provides the ability for crucial electrostatic interaction for vesicle fusion or selective interaction with membrane-related signaling molecules and polypeptides in a synthetically tractable manner. The results obtained may help to develop new approaches for rational design of extremophilic bolalipid-based liposomes for various applications, including delivery of drugs and vaccines.

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III.

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.

Biomolecular Condensates under Extreme Martian Salt Conditions.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

Liquid droplets of protein LAF1 provide a vehicle to regulate storage of the signaling protein K-Ras4B and its transport to the lipid membrane.

Liquid-liquid phase separation has been shown to promote the formation of functional membraneless organelles involved in various cellular processes, including metabolism, stress response and signal transduction. Protein LAF1 found in P-granules phase separates into liquid-like droplets by patterned electrostatic interactions between acidic and basic tracts in LAF1 and has been used as model system in this study. We show that signaling proteins, such as K-Ras4B, a small GTPase that acts as a molecular switch and regulates many cellular processes including proliferation, apoptosis and cell growth, can colocalize in LAF1 droplets. Colocalization is facilitated by electrostatic interactions between the positively charged polybasic domain of K-Ras4B and the negatively charged motifs of LAF1. The interaction partners B- and C-Raf of K-Ras4B can also be recruited to the liquid droplets. Upon contact with an anionic lipid bilayer membrane, the liquid droplets dissolve and K-Ras4B is released, forming nanoclusters in the lipid membrane. Considering the high tuneability of liquid-liquid phase separation in the cell, the colocalization of signaling proteins and their effector molecules in liquid droplets may provide an additional vehicle for regulating storage and transport of membrane-associated signaling proteins such as K-Ras4B and offer an alternative strategy for high-fidelity signal output.

Exploring the polymorphism, conformational dynamics and function of amyloidogenic peptides and proteins by temperature and pressure modulation.

Our understanding of amyloid structures and the mechanisms by which disease-associated peptides and proteins self-assemble into these fibrillar aggregates, has advanced considerably in recent years. It is also established that amyloid fibrils are generally polymorphic. The molecular structures of the aggregation intermediates and the causes of molecular and structural polymorphism are less understood, however. Such information is mandatory to explain the pathological diversity of amyloid diseases. What is also clear is that not only protein mutations, but also the physiological milieu, i.e. pH, cosolutes, crowding and surface interactions, have an impact on fibril formation. In this minireview, we focus on the effect of the less explored physical parameters temperature and pressure on the fibrillization propensity of proteins and how these variables can be used to reveal additional mechanistic information about intermediate states of fibril formation and molecular and structural polymorphism. Generally, amyloids are very stable and can resist harsh environmental conditions, such as extreme pH, high temperature and high pressure, and can hence serve as valuable functional amyloid. As an example, we discuss the effect of temperature and pressure on the catalytic activity of peptide amyloid fibrils that exhibit enzymatic activity.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

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.

Characterization of the Spatial Organization of Raf Isoforms Interacting with K-Ras4B in the Lipid Membrane.

Activation of Raf kinases by the membrane-anchored protein K-Ras4B is a key step of cellular signal regulation. As a predominant variant of the Ras family, K-Ras4B has been considered to be a major drug target in cancer therapy. Therefore, an integrated study of Raf interaction with membrane-associated K-Ras4B is essential. While the Ras-binding domain (RBD) of Raf contains the main binding interface to K-Ras4B, its cysteine-rich domain (CRD) is thought to be responsible for its association with the membrane interface. We applied time-lapse tapping-mode atomic force microscopy to visualize and characterize the interaction of these binding motifs of A-, B-, and C-Raf isoforms with K-Ras4B in a raft-like anionic model biomembrane. However, we found that the RBDs of the Raf isomers are readily recruited to K-Ras4B nanoclusters in the lipid membrane, with different efficiencies. Unexpectedly and different from A-Raf-RBD, B- and C-Raf-RBD are able to bind markedly also directly to the lipid membrane. We also found that Raf-RBD-CRD is readily recruited to the K-Ras4B forming nanoclusters in the fluid membrane phase, with the CRD domains binding to the lipid interface. The K-Ras4B-nanoclusters are likely to enhance Raf binding and activate signaling by enriching the Raf proteins and facilitating formation of Raf dimers. Interestingly, A-, B-, and C-Raf-RBD-CRD are also able to bind directly to the heterogeneous membrane surrounding the K-Ras4B nanoclusters, which could potentially enhance the overall affinity to K-Ras4B in a Raf-isoform-dependent manner. Overall, these results provide new insights into the spatial organization of the membrane-associated Raf-Ras signaling module for the various Raf isoforms, which is important for understanding the activation of Raf kinases and required for the development of drugs against cancers through targeting Raf-Ras interactions.

Interaction of imidazolium-based lipids with phospholipid bilayer membranes of different complexity.

In recent years, alkylated imidazolium salts have been shown to affect lipid membranes and exhibit general cytotoxicity as well as significant anti-tumor activity. Here, we examined the interactions of a sterically demanding, biophysically unexplored imidazolium salt, 1,3-bis(2,6-diisopropylphenyl)-4,5-diundecylimidazolium bromide (C11IPr), on the physico-chemical properties of various model biomembrane systems. The results are compared with those for the smaller headgroup variant 1,3-dimethyl-4,5-diundecylimidazolium iodide (C11IMe). We studied the influence of these two lipid-based imidazolium salts at concentrations from 1 to about 10 mol% on model biomembrane systems of different complexity, including anionic heterogeneous raft membranes which are closer to natural membranes. Fluorescence spectroscopic, DSC, surface potential and FTIR measurements were carried out to reveal changes in membrane thermotropic phase behavior, lipid conformational order, fluidity and headgroup charge. Complementary AFM and confocal fluorescence microscopy measurements allowed us to detect changes in the lateral organization and membrane morphology. Both lipidated imidazolium salts increase the membrane fluidity and lead to a deterioration of the lateral domain structure of the membrane, in particular for C11IPr owing to its bulkier headgroup. Moreover, partitioning of the lipidated imidazolium salts into the lipid vesicles leads to marked changes in lateral organization, curvature and morphology of the lipid vesicles at high concentrations, with C11IPr having a more pronounced effect than C11IMe. Hence, these compounds seem to be vastly suitable for biochemical and biotechnological engineering, with high potentials for antimicrobial activity, drug delivery and gene transfer.

The small molecule inhibitor anle145c thermodynamically traps human islet amyloid peptide in the form of non-cytotoxic oligomers.

Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM.

A hydroxylamine probe for profiling S-acylated fatty acids on proteins.

Reversible S-palmitoylation is a key regulatory mechanism of protein function and localization. There is increasing evidence that S-acylation is not restricted to palmitate but it includes shorter, longer, and unsaturated fatty acids. However, the diversity of this protein modification has not been fully explored. Herein, we report a chemical probe that combined with MS-based analysis allows the rapid detection and quantification of fatty acids linked to proteins. We have used this approach to profile the S-acylome and to show that the oncogene N-Ras is heterogeneously acylated with palmitate and palmitoleate. Studies on protein distribution in membrane subdomains with semisynthetic proteins revealed that unsaturated N-Ras presents an increased tendency toward clustering and higher insertion kinetic rate constants.

The pressure and temperature perturbation approach reveals a whole variety of conformational substates of amyloidogenic hIAPP monitored by 2D NMR spectroscopy.

The intrinsically disordered human islet amyloid polypeptide (hIAPP) is a 37 amino acid peptide hormone that is secreted by pancreatic beta cells along with glucagon and insulin. The glucose metabolism of humans is regulated by a balanced ratio of insulin and hIAPP. The disturbance of this balance can result in the development of type-2 diabetes mellitus (T2DM), whose pathogeny is associated by self-assembly induced aggregation and amyloid deposits of hIAPP into nanofibrils. Here, we report pressure- and temperature-induced changes of NMR chemical shifts of monomeric hIAPP in bulk solution to elucidate the contribution of conformational substates in a residue-specific manner in their role as molecular determinants for the initial self-assembly. The comparison with a similar peptide, the Alzheimer peptide Aß(1-40), which is leading to self-assembly induced aggregation and amyloid deposits as well, reveals that in both peptides highly homologous areas exist (Q10-|L16 and N21-L27 in hIAPP and Q15-A21 and S26-I32 in Aß). The N-terminal area of hIAPP around amino acid residues 3-20 displays large differences in pressure sensitivity compared to Aß, pinpointing to a different structural ensemble in this sequence element which is of helical origin in hIAPP. Knowledge of the structural nature of the highly amyloidogenic hIAPP and the differences with respect to the conformational ensemble of Aß(1-40) will help to identify molecular determinants of self-assembly as well as cross-seeded assembly initiated aggregation and help facilitate the rational design of drugs for therapeutic use.

Effects of in vivo conditions on amyloid aggregation.

One of the grand challenges of biophysical chemistry is to understand the principles that govern protein misfolding and aggregation, which is a highly complex process that is sensitive to initial conditions, operates on a huge range of length- and timescales, and has products that range from protein dimers to macroscopic amyloid fibrils. Aberrant aggregation is associated with more than 25 diseases, which include Alzheimer's, Parkinson's, Huntington's, and type II diabetes. Amyloid aggregation has been extensively studied in the test tube, therefore under conditions that are far from physiological relevance. Hence, there is dire need to extend these investigations to in vivo conditions where amyloid formation is affected by a myriad of biochemical interactions. As a hallmark of neurodegenerative diseases, these interactions need to be understood in detail to develop novel therapeutic interventions, as millions of people globally suffer from neurodegenerative disorders and type II diabetes. The aim of this review is to document the progress in the research on amyloid formation from a physicochemical perspective with a special focus on the physiological factors influencing the aggregation of the amyloid-ß peptide, the islet amyloid polypeptide, α-synuclein, and the hungingtin protein.

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis.

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), 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 new biological function of GRAMD1A and a new role for cholesterol in autophagy.

Dissociation of the Signaling Protein K-Ras4B from Lipid Membranes Induced by a Molecular Tweezer.

Oncogenic Ras mutations occur in more than 30 % of human cancers. K-Ras4B is the most frequently mutated isoform of Ras proteins. Development of effective K-Ras4B inhibitors has been challenging, hence new approaches to inhibit this oncogenic protein are urgently required. The polybasic domain of K-Ras4B with its stretch of lysine residues is essential for its plasma membrane targeting and localization. Employing CD and fluorescence spectroscopy, confocal fluorescence, and atomic force microscopy we show that the molecular tweezer CLR01 is able to efficiently bind to the lysine stretch in the polybasic domain of K-Ras4B, resulting in dissociation of the K-Ras4B protein from the lipid membrane and disintegration of K-Ras4B nanoclusters in the lipid bilayer. These results suggest that targeting of the polybasic domain of K-Ras4B by properly designed tweezers might represent an effective strategy for inactivation of K-Ras4B signaling.