Leveraging a Phage-Encoded Noncanonical Amino Acid: A Novel Pathway to Potent and Selective Epigenetic Reader Protein Inhibitors.

Epigenetic reader proteins interpret histone epigenetic marks to regulate gene expression. Given their vital roles and the link between their dysfunction and various diseases, these proteins present compelling targets for therapeutic interventions. Nevertheless, designing selective inhibitors for these proteins poses significant challenges, primarily due to their unique properties such as shallow binding sites and similarities with homologous proteins. To overcome these challenges, we propose an innovative strategy that uses phage display with a genetically encoded noncanonical amino acid (ncAA) containing an epigenetic mark. This ncAA guides binding to the reader protein's active site, allowing the identification of peptide inhibitors with enhanced affinity and selectivity. In this study, we demonstrate this novel approach's effectiveness by identifying potent inhibitors for the ENL YEATS domain that plays a critical role in leukemogenesis. Our strategy involved genetically incorporating Nε-butyryl-l-lysine (BuK), known for its binding to ENL YEATS, into a phage display library for enriching the pool of potent inhibitors. One resultant hit was further optimized by substituting BuK with other pharmacophores to exploit a unique π-π-π stacking interaction with ENL YEATS. This led to the creation of selective ENL YEATS inhibitors with a KD value of 2.0 nM and a selectivity 28 times higher for ENL YEATS than its close homologue AF9 YEATS. One such inhibitor, tENL-S1f, demonstrated robust cellular target engagement and on-target effects to inhibit leukemia cell growth and suppress the expression of ENL target genes. As a pioneering study, this work opens up extensive avenues for the development of potent and selective peptidyl inhibitors for a broad spectrum of epigenetic reader proteins.

BRD4354 Is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease.

Numerous organic molecules are known to inhibit the main protease (MPro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of MPro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of MPro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against MPro with an IC50 (concentration that inhibits activity by 50%) of 0.72 ± 0.04 µM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a KI of 1.9 ± 0.5 µM followed by a second slow inactivation step, kinact,max of 0.040 ± 0.002 min-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of MPro. Based on these data, a Michael-addition reaction mechanism between MPro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

A Novel Y-Shaped, S-O-N-O-S-Bridged Cross-Link between Three Residues C22, C44, and K61 Is Frequently Observed in the SARS-CoV-2 Main Protease.

As the COVID-19 pathogen, SARS-CoV-2 relies on its main protease (MPro) for pathogenesis and replication. During crystallographic analyses of MPro crystals that were exposed to the air, a uniquely Y-shaped, S-O-N-O-S-bridged post-translational cross-link that connects three residues C22, C44, and K61 at their side chains was frequently observed. As a novel covalent modification, this cross-link serves potentially as a redox switch to regulate the catalytic activity of MPro, a demonstrated drug target of COVID-19. The formation of this linkage leads to a much more open active site that can potentially be targeted for the development of novel SARS-CoV-2 antivirals. The structural rearrangement of MPro by this cross-link indicates that small molecules that lock MPro in the cross-linked form can potentially be used with other active-site-targeting molecules such as paxlovid for synergistic effects in inhibiting SARS-CoV-2 viral replication.

Ratiometric Zinc Biosensor Based on Bioluminescence Resonance Energy Transfer: Trace Metal Ion Determination with Tunable Response.

Determination of metal ions such as zinc in solution remains an important task in analytical and biological chemistry. We describe a novel zinc ion biosensing approach using a carbonic anhydrase-Oplophorus luciferase fusion protein that employs bioluminescence resonance energy transfer (BRET) to transduce the level of free zinc as a ratio of emission intensities in the blue and orange portions of the spectrum. In addition to high sensitivity (below nanomolar levels) and selectivity, this approach allows both quantitative determination of "free" zinc ion (also termed "mobile" or "labile") using bioluminescence ratios and determination of the presence of the ion above a threshold simply by the change in color of bioluminescence, without an instrument. The carbonic anhydrase metal ion sensing platform offers well-established flexibility in sensitivity, selectivity, and response kinetics. Finally, bioluminescence labeling has proven an effective approach for molecular imaging in vivo since no exciting light is required; the expressible nature of this sensor offers the prospect of imaging zinc fluxes in vivo.

Phage-assisted, active site-directed ligand evolution of a potent and selective histone deacetylase 8 inhibitor.

Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+ -dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved N𝜀 -acetyl-lysyl-tRNA synthetase in combination with tRNAPyl , we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+ -chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8-inhibition Ki value of 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01Δ1Δ7 that has an HDAC8-inhibition Ki value of 0.31 nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.

Gambogic acid and juglone inhibit RNase P through distinct mechanisms.

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 µM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates.

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as ɑ-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of EGFR/Akt pathway activation. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.

Combining Active Carbonic Anhydrase with Nanogels: Enzyme Protection and Zinc Sensing.

BACKGROUND: Due to its excellent biocompatibility, the polyacrylamide (PAAm) hydrogel has shown great potential for the immobilization of enzymes used in biomedical applications. The major challenge involved is to preserve, during the immobilization process, both the biological activity and the structural integrity of the enzymes. Here we report, for the first time, a proof-of-concept study for embedding active carbonic anhydrase (CA) into polyacrylamide (PAAm) nanogels. By immobilizing CA in these nanogels, we hope to provide important advantages, such as matrix protection of the CA as well as its targeted delivery, and also for potentially using these nanogels as zinc nano-biosensors, both in-vitro and in-vivo. METHODS AND RESULTS: Two methods are reported here for CA immobilization: encapsulation and surface conjugation. In the encapsulation method, the common process was improved, so as to best preserve the CA, by 1) using a novel biofriendly nonionic surfactant system (Span 80/Tween 80/Brij 30) and 2) using an Al2O3 adsorptive filtration purification procedure. In the surface conjugation method, blank PAAm nanogels were activated by N-hydroxysuccinimide and the CA was cross-linked to the nanogels. The amount of active CA immobilized in the nanoparticles was quantified for both methods. Per 1 g nanogels, the CA encapsulated nanogels contain 11.3 mg active CA, while the CA conjugated nanogels contain 22.5 mg active CA. Also, the CA conjugated nanoparticles successfully measured free Zn2+ levels in solution, with the Zn2+ dissociation constant determined to be 9 pM. CONCLUSION: This work demonstrates universal methods for immobilizing highly fragile bio-macromolecules inside nanoparticle carriers, while preserving their structural integrity and biological activity. The advantages and limitations are discussed, as well as the potential biomedical applications.

Disease-associated mutations in mitochondrial precursor tRNAs affect binding, m1R9 methylation, and tRNA processing by mtRNase P.

Mitochondrial diseases linked to mutations in mitochondrial (mt) tRNA sequences are common. However, the contributions of these tRNA mutations to the development of diseases is mostly unknown. Mutations may affect interactions with (mt)tRNA maturation enzymes or protein synthesis machinery leading to mitochondrial dysfunction. In human mitochondria, in most cases the first step of tRNA processing is the removal of the 5' leader of precursor tRNAs (pre-tRNA) catalyzed by the three-component enzyme, mtRNase P. Additionally, one component of mtRNase P, mitochondrial RNase P protein 1 (MRPP1), catalyzes methylation of the R9 base in pre-tRNAs. Despite the central role of 5' end processing in mitochondrial tRNA maturation, the link between mtRNase P and diseases is mostly unexplored. Here, we investigate how 11 different human disease-linked mutations in (mt)pre-tRNAIle, (mt)pre-tRNALeu(UUR), and (mt)pre-tRNAMet affect the activities of mtRNase P. We find that several mutations weaken the pre-tRNA binding affinity (KD s are approximately two- to sixfold higher than that of wild-type), while the majority of mutations decrease 5' end processing and methylation activity catalyzed by mtRNase P (up to ∼55% and 90% reduction, respectively). Furthermore, all of the investigated mutations in (mt)pre-tRNALeu(UUR) alter the tRNA fold which contributes to the partial loss of function of mtRNase P. Overall, these results reveal an etiological link between early steps of (mt)tRNA-substrate processing and mitochondrial disease.

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors*.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.

Unexpected specificity within dynamic transcriptional protein-protein complexes.

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator-not often considered as contributing to binding-play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

A Speedy Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M Pro ) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M Pro ), we have designed and synthesized a series of SC2M Pro inhibitors that contain ß-( S -2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M Pro active site cysteine C145. All inhibitors display high potency with IC 50 values at or below 100 nM. The most potent compound MPI3 has as an IC 50 value as 8.5 nM. Crystallographic analyses of SC2M Pro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M Pro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA.

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5'-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-ß Generates Toxic Hetero-oligomers.

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and ß-amyloid (Aß) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aß(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aß(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aß(M1-42) heteromers comprised of unstructured Aß(M1-42) and helical 4F. A uniform two-fold reduction in 15N/1H NMR signal intensities of Aß(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aß(M1-42) is electrostatically driven and induces unfolding of Aß(M1-42). Neurotoxicity profiling of Aß(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aß(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant.

Histone deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of kcat/KM for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ∼10-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat, revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency, and substrate selectivity.

Interplay between substrate recognition, 5' end tRNA processing and methylation activity of human mitochondrial RNase P.

Human mitochondrial ribonuclease P (mtRNase P) is an essential three-protein complex that catalyzes the 5' end maturation of mitochondrial precursor tRNAs (pre-tRNAs). Mitochondrial RNase P Protein 3 (MRPP3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl-methionine (SAM)-dependent methyltransferase MRPP1/2 subcomplex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here, we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the TRMT10C (MRPP1) cofactor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process, and methylate human mitochondrial pre-tRNAs.

Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

The chaperone SmgGDS-607 has a dual role, both activating and inhibiting farnesylation of small GTPases.

Ras family small GTPases undergo prenylation (such as farnesylation) for proper localization to the plasma membrane, where they can initiate oncogenic signaling pathways. Small GTP-binding protein GDP-dissociation stimulator (SmgGDS) proteins are chaperones that bind and traffic small GTPases, although their exact cellular function is unknown. Initially, SmgGDS proteins were classified as guanine nucleotide exchange factors, but recent findings suggest that SmgGDS proteins also regulate prenylation of small GTPases in vivo in a substrate-selective manner. SmgGDS-607 recognizes the polybasic region and the CAAX box of several small GTPases and inhibits prenylation by impeding their entry into the geranylgeranylation pathway. Here, using recombinant and purified enzymes for prenylation and protein-binding assays, we demonstrate that SmgGDS-607 differentially regulates farnesylation of several small GTPases. SmgGDS-607 inhibited farnesylation of some proteins, such as DiRas1, by sequestering the protein and limiting modification catalyzed by protein farnesyltransferase (FTase). We found that the competitive binding affinities of the small GTPase for SmgGDS-607 and FTase dictate the extent of this inhibition. Additionally, we discovered that SmgGDS-607 increases the rate of farnesylation of HRas by enhancing product release from FTase. Our work indicates that SmgGDS-607 binds to a broad range of small GTPases and does not require a PBR for recognition. Together, these results provide mechanistic insight into SmgGDS-607-mediated regulation of farnesylation of small GTPases and suggest that SmgGDS-607 has multiple modes of substrate recognition.

A cationic polymethacrylate-copolymer acts as an agonist for ß-amyloid and an antagonist for amylin fibrillation.

In humans, ß-amyloid and islet amyloid polypeptide (IAPP, also known as amylin) aggregations are linked to Alzheimer's disease and type-2 diabetes, respectively. There is significant interest in better understanding the aggregation process by using chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce a ß-hairpin structure and accelerate the formation of amorphous aggregates of ß-amyloid-1-40, whereas it constrains the conformational plasticity of amylin for several days and slows down its aggregation at substoichiometric polymer concentrations. NMR experiments and microsecond scale atomistic molecular dynamics simulations reveal that PMAQA interacts with ß-amyloid-1-40 residues spanning regions K16-V24 and A30-V40 followed by ß-sheet induction. For amylin, it binds strongly close to the amyloid core domain (NFGAIL) and restrains its structural rearrangement. High-speed atomic force microscopy and transmission electron microscopy experiments show that PMAQA blocks the nucleation and fibrillation of amylin, whereas it induces the formation of amorphous aggregates of ß-amyloid-1-40. Thus, the reported study provides a valuable approach to develop polymer-based amyloid inhibitors to suppress the formation of toxic intermediates of ß-amyloid-1-40 and amylin.

Alzheimer's amyloid-beta intermediates generated using polymer-nanodiscs.

Polymethacrylate-copolymer (PMA) encased lipid-nanodiscs (∼10 nm) and macro-nanodiscs (>15 nm) are used to study Aß1-40 aggregation. We demonstrate that PMA-nanodiscs form a ternary association with Aß and regulate its aggregation kinetics by trapping intermediates. Results demonstrating the reduced neurotoxicity of nanodisc-bound Aß oligomers are also reported.