Aggregation Pathway Research Articles

Intrinsic conformational preference in the monomeric protein governs amyloid polymorphism.

The inherent stochasticity associated with the hierarchical self-assembly of either native-like or partially-unfolded protein monomers leads to the formation of transient, morphologically-diverse prefibrillar species resulting in structurally-distinct polymorphic protein aggregates. High-resolution structural characterization of mature aggregates has revealed heterogeneous supramolecular packing of protofibrils within amyloid polymorphs. However, little is known about whether initial monomeric protein conformers engender polymorphism at the onset of aggregation. Here, we show that intrinsic conformational preference in aggregation-competent monomeric ovalbumin, an archetypal serpin, dictates fibrillar polymorphism by modulating aggregation pathways. Using fluorescence, FT-IR, and vibrational Raman spectroscopy coupled with dynamic light scattering and electron microscopy, we demonstrate that conformationally-diverse amyloidogenic monomers, formed via an interplay of electrostatic and hydrophobic interactions before the commencement of aggregation, play a crucial role in promoting amyloid polymorphism. Moreover, the monomeric conformational fingerprints, accrued at the onset of aggregation, persist and propagate during the formation of polymorphic amyloids. Our results delineate essential conformational characteristics of the monomeric protein preceding aggregation, which will have broad implications in the mechanistic understanding of amyloid strain diversity observed in disease-related proteins.

Non-fibril amyloid aggregation at the air/water interface: self-adaptive pathway resulting in a 2D Janus nanofilm.

The amyloid states of proteins are implicated in several neurodegenerative diseases and bioadhesion processes. However, the classical amyloid fibrillization mechanism fails to adequately explain the formation of polymorphic aggregates and their adhesion to various surfaces. Herein, we report a non-fibril amyloid aggregation pathway, with disulfide-bond-reduced lysozyme (R-Lyz) as a model protein under quasi-physiological conditions. Very different from classical fibrillization, this pathway begins with the air-water interface (AWI) accelerated oligomerization of unfolded full-length protein, resulting in unique plate-like oligomers with self-adaptive ability, which can adjust their conformations to match various interfaces such as the asymmetric AWI and amyloid-protein film surface. The pathway enables a stepwise packing of the plate-like oligomers into a 2D Janus nanofilm, exhibiting a divergent distribution of hydrophilic/hydrophobic residues on opposite sides of the nanofilm. The resulting Janus nanofilm possesses a top-level Young's modulus (8.3 ± 0.6 GPa) among amyloid-based materials and exhibits adhesive strength two times higher (145 ± 81 kPa) than that of barnacle cement. Furthermore, we found that such an interface-directed pathway exists in several amyloidogenic proteins with a similar self-adaptive 2D-aggregation process, including bovine serum albumin, insulin, fibrinogen, hemoglobin, lactoferrin, and ovalbumin. Thus, our findings on the non-fibril self-adaptive mechanism for amyloid aggregation may shed light on polymorphic amyloid assembly and their adhesions through an alternative pathway.

Each big journey starts with a first step: Importance of oligomerization.

Protein oligomers, widely found in nature, have significant physiological and pathological functions. They are classified into three groups based on their function and toxicity. Significant advancements are being achieved in the development of functional oligomers, with a focus on various applications and their engineering. The antimicrobial peptides oligomers play roles in death of bacterial and cancer cells. The predominant pathogenic species in neurodegenerative disorders, as shown by recent results, are amyloid oligomers, which are the main subject of this chapter. They are generated throughout the aggregation process, serving as both intermediates in the subsequent aggregation pathways and ultimate products. Some of them may possess potent cytotoxic properties and through diverse mechanisms cause cellular impairment, and ultimately, the death of cells and disease progression. Information regarding their structure, formation mechanism, and toxicity is limited due to their inherent instability and structural variability. This chapter aims to provide a concise overview of the current knowledge regarding amyloid oligomers.

Computational investigation of copper-mediated conformational changes in α-synuclein dimer.

We report molecular dynamics simulation of dimers of α-synuclein, the peptide closely associated with onset of Parkinson's disease, both as metal-free dimer and with inter-chain bridging provided by Cu(II) ions. Our investigation reveals that the presence of copper-induced inter-chain bridging not only stabilizes α-synuclein dimers, but also leads to enhanced β-sheet formation at critical regions within the N-terminal and NAC regions of the protein. These contacts are larger and longer-lived in the presence of copper, and as a result each peptide chain is more extended and less flexible than in the metal-free dimer. The persistence of these inter-peptide contacts underscores their significance in stabilising the dimers, potentially influencing the aggregation pathway. Moreover, the increased flexibility in the two termini, as well as the absence of persistent contacts in the metal-free dimer, correlates with the presence of amorphous aggregates. This phenomenon is known to mitigate fibrillation, while their absence in the metal-bound dimer suggests an increased propensity to form fibrils in the presence of copper ions.

Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors.

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C18 column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bβ and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.

Fluorescent protein tagging promotes phase separation and alters the aggregation pathway of huntingtin exon-1

Fluorescent protein tags are convenient tools for tracking the aggregation states of amyloidogenic or phase separating proteins, but the effect of the tags is often not well understood. Here, we investigated the impact of a C-terminal red fluorescent protein (RFP) tag on the phase separation of huntingtin exon-1 (Httex1), an N-terminal portion of the huntingtin protein that aggregates in Huntington's disease. We found that the RFP-tagged Httex1 rapidly formed micron-sized, phase separated states in the presence of a crowding agent. The formed structures had a rounded appearance and were highly dynamic according to electron paramagnetic resonance and fluorescence recovery after photobleaching, suggesting that the phase separated state was largely liquid in nature. Remarkably, the untagged protein did not undergo any detectable liquid condensate formation under the same conditions. In addition to strongly promoting liquid-liquid phase separation, the RFP tag also facilitated fibril formation, as the tag-dependent liquid condensates rapidly underwent a liquid-to-solid transition. The rate of fibril formation under these conditions was significantly faster than that of the untagged protein. When expressed in cells, the RFP-tagged Httex1 formed larger aggregates with different antibody staining patterns compared to untagged Httex1. Collectively, these data reveal that the addition of a fluorescent protein tag significantly impacts liquid and solid phase separations of Httex1 invitro and leads to altered aggregation in cells. Considering that the tagged Httex1 is commonly used to study the mechanisms of Httex1 misfolding and toxicity, our findings highlight the importance to validate the conclusions with untagged protein.

About the pathway of amyloid aggregation of titin

The process of amyloid aggregation is quite complex and poorly understood. In this work, having summarized previously obtained results on the aggregation of the multidomain smooth muscle protein titin, an attempt has been made to expand understanding of this process, and a new possible mechanism by which amyloid aggregation of titin may occur is delineated. Our main conclusion is that the ability of titin to form amorphous aggregates seems to be the only possible way of aggregation of this protein. Most likely, only separate parts of the molecules, but not the whole protein, are involved in the formation of the amyloid structure in amorphous aggregates of smooth muscle titin. This feature, given the large size of the protein molecule, distinguishes titin from other amyloid or amyloid-like proteins. The paper discusses the potential energy landscape underlying the formation of titin amyloid aggregates.

Comparative analysis of peripheral whole blood transcriptome from asymptomatic carriers reveals upregulation of subsets of surface proteins implicated in Plasmodium falciparum phenotypic plasticity

The molecular mechanism underlying Plasmodium falciparum's persistence in the asymptomatic phase of infection remains largely unknown. However, large-scale shifts in the parasites' gene expression during asymptomatic infections may enhance phenotypic plasticity, maximizing their fitness and leading to the persistence of the asymptomatic infections. To uncover these mechanisms, we aimed to identify parasite genetic factors implicated in asymptomatic infections through whole transcriptome analysis. We analyzed publicly available transcriptome datasets containing asymptomatic malaria (ASM), uncomplicated malaria (SM), and malaria-naïve (NSM) samples from 35 subjects for differentially expressed genes (DEGs) and long noncoding RNAs. Our analysis identified 755 and 1773 DEGs in ASM vs SM and NSM, respectively. These DEGs revealed sets of genes coding for proteins of unknown functions (PUFs) upregulated in ASM vs SM and ASM, suggesting their role in underlying fundamental molecular mechanisms during asymptomatic infections. Upregulated genes in ASM vs SM revealed a subset of 24 clonal variant genes (CVGs) involved in host-parasite and symbiotic interactions and modulation of the symbiont of host erythrocyte aggregation pathways. Moreover, we identified 237 differentially expressed noncoding RNAs in ASM vs SM, of which 11 were found to interact with CVGs, suggesting their possible role in regulating the expression of CVGs. Our results suggest that P. falciparum utilizes phenotypic plasticity as an adaptive mechanism during asymptomatic infections by upregulating clonal variant genes, with long noncoding RNAs possibly playing a crucial role in their regulation. Thus, our study provides insights into the parasites' genetic factors that confer a fitness advantage during asymptomatic infections.

Differential expression of PSMC4, SKP1, and HSPA8 in Parkinson's disease: insights from a Mexican mestizo population.

Parkinson's disease (PD) is a complex neurodegenerative condition characterized by alpha-synuclein aggregation and dysfunctional protein degradation pathways. This study investigates the differential gene expression of pivotal components (UBE2K, PSMC4, SKP1, and HSPA8) within these pathways in a Mexican-Mestizo PD population compared to healthy controls. We enrolled 87 PD patients and 87 controls, assessing their gene expression levels via RT-qPCR. Our results reveal a significant downregulation of PSMC4, SKP1, and HSPA8 in the PD group (p = 0.033, p = 0.003, and p = 0.002, respectively). Logistic regression analyses establish a strong association between PD and reduced expression of PSMC4, SKP1, and HSPA8 (OR = 0.640, 95% CI = 0.415-0.987; OR = 0.000, 95% CI = 0.000-0.075; OR = 0.550, 95% CI = 0.368-0.823, respectively). Conversely, UBE2K exhibited no significant association or expression difference between the groups. Furthermore, we develop a gene expression model based on HSPA8, PSMC4, and SKP1, demonstrating robust discrimination between healthy controls and PD patients. Notably, the model's diagnostic efficacy is particularly pronounced in early-stage PD. In conclusion, our study provides compelling evidence linking decreased gene expression of PSMC4, SKP1, and HSPA8 to PD in the Mexican-Mestizo population. Additionally, our gene expression model exhibits promise as a diagnostic tool, particularly for early-stage PD diagnosis.

The Pathway of Amyloid Aggregation of Titin

The Pathway of Amyloid Aggregation of Titin

Understanding the electrochemical interaction between the hybrid bilayer lipid membranes and amyloid‐β

AbstractBackgroundBiomimetic membrane systems can be deposited onto conductive metal surfaces to study the physical properties of membranes as well as the behaviour of proteins within these membranes using electrochemical and surface analytical techniques [1]. Although there are several types of biomimetic membrane, hybrid bilayer lipid membranes (HBLMs) have attracted wide interest. Traditionally, HBLMs have been developed by electrodepositing alkanethiol self‐assembled monolayers onto gold surfaces, which can then be further modified by depositing a secondary phospholipid layer to form the HBLM. Although HBLMs have been widely regarded due to them being robust, relatively straightforward to make, as well as being easy to modify according to the users needs, several concerns surround HBLMs including their reduced fluidity compared to true cell membranes. This is due to the alkanethiol layer being crystalline in nature and more densely packed, resulting in difficulty to reconstitute transmembrane proteins within them.MethodScreen‐printed carbon electrodes are modified with diazonium based phenyl alkyl groups using different protocols to evaluate the biomimetic differences of these novel HBLMs. 2,2‐diphenyl‐1‐picrylhydrazyl is used to prevent the formation of polyaryl layers on the surface of the electrode, hence limiting the electrodeposition to a monolayer. The HBLMs are formed by depositing the second layer in the form of dihexadecyl phosphate, which are analyzed using differential pulse voltammetry, ellipsometry, and X‐ray photoelectron spectroscopy.ResultThe different HBLMs presented are embedded with amyloid‐β1‐42 (Aβ42), the hallmark protein implicated in Alzheimer’s Disease, to assess the protein‐membrane interactions using HBLMs. Aβ42 is known to aggregate to form dimers, oligomers, fibrils, and plaques. Hence using electrochemical techniques, we are able to monitor the aggregation of Aβ42 in the HBLMs. The aggregation process was accelerated by the presence of the bilayer membrane.ConclusionThe electrochemical platform presented here enables us to understand how the interaction of amyloid‐β with the bilayer membrane can alter the aggregation pathways and also affect the formation of toxic oligomer species.

Nanoplastic Stimulates the Amyloidogenesis of Parkinson's Alpha-Synuclein NACore.

Environmental plastic wastes are potential health hazards due to their prevalence as well as their versatility in initiating physical, chemical, and biological interactions and transformations. Indeed, recent research has implicated the adverse effects of micro- and nano-plastics, including their neurotoxicity, yet how plastic particulates may impact the aggregation pathway and toxicity of amyloid proteins pertinent to the pathologies of neurological diseases remains unknown. Here, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) is employed to reveal the polymorphic oligomerization of NACore, a surrogate of alpha-synuclein that is associated with the pathogenesis of Parkinson's disease. These data indicate that the production rate and population of the NACore oligomers are modulated by their exposure to a polystyrene nanoplastic, and these cellular assays further reveal an elevated NACore toxicity in microglial cells elicited by the nanoplastic. These simulations confirm that the nanoplastic-NACore association is promoted by their hydrophobic interactions. These findings are corroborated by an impairment in zebrafish hatching, survival, and development in vivo upon their embryonic exposure to the nanoplastic. Together, this study has uncovered the dynamics and mechanism of amyloidogenesis elevated by a nanoplastic trigger, shedding a new light on the neurological burden of plastic pollution.

Mapping the configurational landscape and aggregation phase behavior of the tau protein fragment PHF6.

The PHF6 (Val-Gln-Ile-Val-Tyr-Lys) motif, found in all isoforms of the microtubule-associated protein tau, forms an integral part of ordered cores of amyloid fibrils formed in tauopathies and is thought to play a fundamental role in tau aggregation. Because PHF6 as an isolated hexapeptide assembles into ordered fibrils on its own, it is investigated as a minimal model for insight into the initial stages of aggregation of larger tau fragments. Even for this small peptide, however, the large length and time scales associated with fibrillization pose challenges for simulation studies of its dynamic assembly, equilibrium configurational landscape, and phase behavior. Here, we develop an accurate, bottom-up coarse-grained model of PHF6 for large-scale simulations of its aggregation, which we use to uncover molecular interactions and thermodynamic driving forces governing its assembly. The model, not trained on any explicit information about fibrillar structure, predicts coexistence of formed fibrils with monomers in solution, and we calculate a putative equilibrium phase diagram in concentration-temperature space. We also characterize the configurational and free energetic landscape of PHF6 oligomers. Importantly, we demonstrate with a model of heparin that this widely studied cofactor enhances the aggregation propensity of PHF6 by ordering monomers during nucleation and remaining associated with growing fibrils, consistent with experimentally characterized heparin-tau interactions. Overall, this effort provides detailed molecular insight into PHF6 aggregation thermodynamics and pathways and, furthermore, demonstrates the potential of modern multiscale modeling techniques to produce predictive models of amyloidogenic peptides simultaneously capturing sequence-specific effects and emergent aggregate structures.

Abstract 13577: Burden of Cardiometabolic Risk Factors and Vascular Health

Introduction: Cardiometabolic risk factors (diabetes, obesity, and hypertension) are highly prevalent and contribute to cardiovascular disease (CVD). Endothelial dysfunction often precedes CVD development. Direct brachial vein endothelial cell (EC) harvesting enables the examination of vascular health via assessment of EC gene expression. Hypothesis: We hypothesized that individuals with greater cardiometabolic risk would demonstrate a more pro-inflammatory EC transcriptome. Methods: Adult participants without CVD underwent EC harvesting followed by RNA sequencing. We categorized individuals by number of risk factors (hypertension, diabetes, obesity) as 0 through 3. To evaluate the association between risk factor burden and outcome transcripts, we performed multivariable linear regression, adjusting for age, sex, and race/ethnicity. Results: Among 18 individuals (mean age 47 ± 14, 44% female, and 61% white), 17%, 44%, 28%, and 11% had 0,1,2, and 3 prevalent risk factors, respectively. EC RNA sequencing revealed 588 differentially expressed transcripts (p-adj <0.05) with 585 upregulated, 3 downregulated, and excellent clustering ( Fig. a, b ). Gene ontology enrichment analysis demonstrated enriched and upregulated pathways of T-cell activation (NES = 2.22, p<0.001), leukocyte differentiation (NES= 2.16, p<0.001), and platelet aggregation (NES= 1.66, p=0.03) and downregulated pathways of endothelial cell proliferation (NES= -1.68, p=0.03). ( Fig. c ) Select upregulated genes (log 2FC >3, p-adj<0.05) included VCAM1, CEACAM1, ADAM 17, and CD99L2, with a graded increase in mean normalized counts with increasing number of risk factors. ( Fig. d ) Conclusions: We demonstrate a proinflammatory, pro-adhesive, and pro-coagulant EC transcriptome associated with increased cardiometabolic risk factor burden offering insight into potential mechanisms linking cardiometabolic risk factors with the development of CVD.

Modulation of Alpha-Synuclein Conformational Ensemble and Aggregation Pathways by Dopamine and Related Molecules.

Dopaminergic neurons are constantly threatened by the thin boundaries between functional α-synuclein (AS) structural disorder and pathogenic aggregation, and between dopamine (DA) neurotransmitter activity and accumulation of cytotoxic by-products. The possibilities of developing drugs for Parkinson's disease (PD) depend on our understanding of the molecular mechanisms that cause or accompany the pathological structural changes in AS. This review focuses on the three interconnected aspects of AS conformational transitions, its aggregation pathways and ligand binding. Specifically, the interactions of AS with DA, DA metabolites, DA analogs and DA agonists are considered. Recent advances in the field are discussed with reference to the structural properties of AS and the methodologies employed. Although several issues are still object of debate, salient structural features of the protein, the aggregates and the ligands can be identified, in the hope of fueling experimental and computational approaches to the discovery of novel disease-modifying agents.

A Missing Origin of the Tau Protein Aggregation Pathway Triggered by Thermal and Biological Forces.

Fluctuations in mechanical force vectors within living cells can substantially influence the behavior and functions of proteins. Tau protein can spontaneously be raptured and entangled in refolding under picoNewton compressive forces that are biologically available in a living cell: a hidden aggregation pathway due to stress and crowding. Our findings were achieved through a customized modification of atomic force microscopy (AFM) for single-molecule manipulation. This previously hidden phenomenon of proteins rupturing collectively while subsequently and spontaneously refolding into a complex entangled conformation, distinct from the Tau protein's folded or unfolded states, could potentially explain the early-event initiation of the aggregation of the Tau protein seen in various neurodegenerative diseases. This article introduces our recent discovery of the missing Tau protein property that is of significant relevance to the Tau protein and neurodegenerative disease research and medical treatment, aiming to stimulate the collective observation and a new perspective on the Tau aggregation mechanism and disease mechanism studies.

Native and Non-Native aggregation pathways of antibodies anticipated by cold-accelerated studies

Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage. In this work, we measured the aggregation rates of two antibodies in a broad temperature range (from 60 °C to −25 °C), using an isochoric cooling method to prevent freezing of the formulations below 0 °C. Both antibodies evidenced increasing aggregation rates when approaching extreme temperatures, because of hot and cold denaturation. This behavior was modelled using Arrhenius and Gibbs-Helmholtz equations, which enabled to deconvolute the contribution of unfolding from the protein association kinetics. This approach made possible to model the aggregation rates at refrigeration temperature (5 °C) in a relatively short timeframe (1–2 weeks) and using standard characterization techniques (SEC-HPLC and DLS).

The Cellular Environment Guides Self‐Assembly and Structural Conformations of Microtubule‐Associated Protein Tau (MAPT)

AbstractIn neurodegenerative tauopathies, such as Alzheimer's disease (AD), microtubule‐associated protein tau (MAPT/tau) transitions from a soluble form to insoluble, filamentous lesions inside affected neurons. During this process, tau adopts a range of physical configurations: from misfolded monomers to higher‐order oligomers and fibrils. Tau aggregation is also associated with changes in post‐translational modifications (PTMs), such as ubiquitination, oxidation, glycation, hyper‐phosphorylation and acetylation, which collectively produce an impressive range of possible tau proteoforms. Many of these tau proteoforms are highly cationic and unlikely to self‐assemble without neutralization of their charges. Indeed, tau fibrils from patients contain anionic biomacromolecules and bound proteins, suggesting that cytosolic components contribute to fibrilligenesis. Here, we review what is known about how the cytosol impacts tau's aggregation pathways. We also speculate that the composition of each brain region (e. g., redox state, tau proteoforms, levels of permissive polyanions, etc.) might play an active role in shaping the structure of the resulting tau fibrils. Although much remains to be discovered, a greater understanding of the role of the cytosol on tau self‐assembly might lead to identification of new therapeutic targets.

Effect of Bacterial Amyloid Protein Phenol-Soluble Modulin Alpha 3 on the Aggregation of Amyloid Beta Protein Associated with Alzheimer's Disease.

Since the proposal of the brainstem axis theory, increasing research attention has been paid to the interactions between bacterial amyloids produced by intestinal flora and the amyloid β-protein (Aβ) related to Alzheimer's disease (AD), and it has been considered as the possible cause of AD. Therefore, phenol-soluble modulin (PSM) α3, the most virulent protein secreted by Staphylococcus aureus, has attracted much attention. In this work, the effect of PSMα3 with a unique cross-α fibril architecture on the aggregation of pathogenic Aβ40 of AD was studied by extensive biophysical characterizations. The results proposed that the PSMα3 monomer inhibited the aggregation of Aβ40 in a concentration-dependent manner and changed the aggregation pathway to form granular aggregates. However, PSMα3 oligomers promoted the generation of the β-sheet structure, thus shortening the lag phase of Aβ40 aggregation. Moreover, the higher the cross-α content of PSMα3, the stronger the effect of the promotion, indicating that the cross-α structure of PSMα3 plays a crucial role in the aggregation of Aβ40. Further molecular dynamics (MD) simulations have shown that the Met1-Gly20 region in the PSMα3 monomer can be combined with the Asp1-Ala2 and His13-Val36 regions in the Aβ40 monomer by hydrophobic and electrostatic interactions, which prevents the conformational conversion of Aβ40 from the α-helix to β-sheet structure. By contrast, PSMα3 oligomers mainly combined with the central hydrophobic core (CHC) and the C-terminal region of the Aβ40 monomer by weak H-bonding and hydrophobic interactions, which could not inhibit the transition to the β-sheet structure in the aggregation pathway. Thus, the research has unraveled molecular interactions between Aβ40 and PSMα3 of different structures and provided a deeper understanding of the complex interactions between bacterial amyloids and AD-related pathogenic Aβ.

Pathways of hLL-3717-29 Aggregation Give Insight into the Mechanism of α-Amyloid Formation.

α-amyloids present a novel self-assembly principle that can be utilized to prepare functional biomaterials. Evidence of α-amyloid formation in the active core of the human LL-37 protein (comprising residues 17 to 29) was associated with this peptide's membranolytic property. Though mechanistic pathways of β-amyloid formation are known, such studies are scarce in α-amyloids. Modern computational techniques allow such mechanistic studies in molecular detail. Here, we propose aggregation pathways in hLL-3717-29 through molecular dynamics simulations. We first identified oligomers among peptides based on a distance criterion. The distribution of oligomers was then used to build Markov state models from which pathways were obtained using the framework of transition path theory. We checked the structural stability of the peptides during oligomerization, which is crucial from their functional point of view. We also investigated the key residues that participate in oligomer formation, the interactions between them, and the effect of residue mutations on the binding free energy of the peptides. Our findings suggest that larger oligomers are produced from the association of smaller and intermediate oligomers. The peptides retain their helical structure during aggregation with transient occurrences of 3-10 helix and turns. Hydrophobic interactions are vital in the aggregation of these peptides with Ile24 playing a crucial role. Mutation of this residue to alanine decreases the peptides' binding free energy, resulting in reduced aggregation tendency.