Structural Mimicry in Microbial and Antimicrobial Amyloids.

The remarkable variety of microbial species of human pathogens and microbiomes generates significant quantities of secreted amyloids, which are structured protein fibrils that serve diverse functions related to virulence and interactions with the host. Human amyloids are associated largely with fatal neurodegenerative and systemic aggregation diseases, and current research has put forward the hypothesis that the interspecies amyloid interactome has physiological and pathological significance. Moreover, functional and molecular-level connections between antimicrobial activity and amyloid structures suggest a neuroimmune role for amyloids that are otherwise known to be pathological. Compared to the extensive structural information that has been accumulated for human amyloids, high-resolution structures of microbial and antimicrobial amyloids are only emerging. These recent structures reveal both similarities and surprising departures from the typical amyloid motif, in accordance with their diverse activities, and advance the discovery of novel antivirulence and antimicrobial agents. In addition, the structural information has led researchers to postulate that amyloidogenic sequences are natural targets for structural mimicry, for instance in host-microbe interactions. Microbial amyloid research could ultimately be used to fight aggressive infections and possibly processes leading to autoimmune and neurodegenerative diseases.

What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-ß and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated ß-strand sheets forming a cross-ß configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-ß amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.

The amphibian antimicrobial peptide uperin 3.5 is a cross-α/cross-ß chameleon functional amyloid.

Antimicrobial activity is being increasingly linked to amyloid fibril formation, suggesting physiological roles for some human amyloids, which have historically been viewed as strictly pathological agents. This work reports on formation of functional cross-α amyloid fibrils of the amphibian antimicrobial peptide uperin 3.5 at atomic resolution, an architecture initially discovered in the bacterial PSMα3 cytotoxin. The fibrils of uperin 3.5 and PSMα3 comprised antiparallel and parallel helical sheets, respectively, recapitulating properties of ß-sheets. Uperin 3.5 demonstrated chameleon properties of a secondary structure switch, forming mostly cross-ß fibrils in the absence of lipids. Uperin 3.5 helical fibril formation was largely induced by, and formed on, bacterial cells or membrane mimetics, and led to membrane damage and cell death. These findings suggest a regulation mechanism, which includes storage of inactive peptides as well as environmentally induced activation of uperin 3.5, via chameleon cross-α/ß amyloid fibrils.

Signal inhibitory receptor on leukocytes-1 recognizes bacterial and endogenous amphipathic α-helical peptides.

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is a negative regulator of myeloid cell function and dampens antimicrobial responses. We here show that different species of the genus Staphylococcus secrete SIRL-1-engaging factors. By screening a library of single-gene transposon mutants in Staphylococcus aureus, we identified these factors as phenol-soluble modulins (PSMs). PSMs are amphipathic α-helical peptides involved in multiple aspects of staphylococcal virulence and physiology. They are cytotoxic and activate the chemotactic formyl peptide receptor 2 (FPR2) on immune cells. Human cathelicidin LL-37 is also an amphipathic α-helical peptide with antimicrobial and chemotactic activities, structurally and functionally similar to α-type PSMs. We demonstrate that α-type PSMs from multiple staphylococcal species as well as human cathelicidin LL-37 activate SIRL-1, suggesting that SIRL-1 recognizes α-helical peptides with an amphipathic arrangement of hydrophobicity, although we were not able to show direct binding to SIRL-1. Upon rational peptide design, we identified artificial peptides in which the capacity to ligate SIRL-1 is segregated from cytotoxic and FPR2-activating properties, allowing specific engagement of SIRL-1. In conclusion, we propose staphylococcal PSMs and human LL-37 as a potential new class of natural ligands for SIRL-1.

Rare by Natural Selection: Disulfide-Bonded Supramolecular Antimicrobial Peptides.

Human LL-3717-29 is an antimicrobial peptide forming thermostable supramolecular fibrils that surround bacterial cells. The crystal structure of LL-3717-29 bearing an I24C substitution of most buried position in the fibril revealed disulfide-bonded dimers that further assembled into a fibrillar structure of densely packed helices. We further demonstrated the position-dependent controllable antibacterial activity of LL-3717-29 I24C and other cysteine mutants, mediated by regulation of intermolecular disulfide bonds and their role in the formation of supramolecular structures. The morphology of the fibrils and their antibacterial mechanism of action might be dependent on their interactions with specific bacteria. The significant effect of disulfide bonds on the assembly into supramolecular structures and their sensitivity to reducing/oxidizing conditions may explain why short helical antimicrobial peptides with a single cysteine and an odd number of cysteines are selected against in nature.

Natural Antimicrobial Peptides Self-assemble as α/ß Chameleon Amyloids.

Amyloid protein fibrils and some antimicrobial peptides (AMPs) share biophysical and structural properties. This observation suggests that ordered self-assembly can act as an AMP-regulating mechanism, and, vice versa, that human amyloids play a role in host defense against pathogens, as opposed to their common association with neurodegenerative and systemic diseases. Based on previous structural information on toxic amyloid peptides, we developed a sequence-based bioinformatics platform and, led by its predictions, experimentally identified 14 fibril-forming AMPs (ffAMPs) from living organisms, which demonstrated cross-ß and cross-α amyloid properties. The results support the amyloid-antimicrobial link. The high prevalence of ffAMPs produced by amphibians and marine creatures among other species suggests that they confer unique advantageous properties in distinctive environments, potentially providing stability and adherence properties. Most of the newly identified 14 ffAMPs showed lipid-induced and/or time-dependent secondary structure transitions in the fibril form, indicating structural and functional cross-α/ß chameleons. Specifically, ffAMP cytotoxicity against human cells correlated with the inherent or lipid-induced α-helical fibril structure. The findings raise hypotheses about the role of fibril secondary structure switching in regulation of processes, such as the transition between a stable storage conformation and an active state with toxicity against specific cell types.

Structural Insights into Curli CsgA Cross-ß Fibril Architecture Inspire Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents.

Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated ß-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer's disease-associated amyloid-ß, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, as previously shown, CsgA fibrils cross-seeded fibrillation of amyloid-ß, providing support for the proposed structural resemblance and potential for cross-species amyloid interactions. The presented findings provide structural insights into amyloidogenic regions important for curli formation, suggest a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases.

Identification of two principal amyloid-driving segments in variable domains of Ig light chains in systemic light-chain amyloidosis.

Systemic light-chain amyloidosis (AL) is a human disease caused by overexpression of monoclonal immunoglobulin light chains that form pathogenic amyloid fibrils. These amyloid fibrils deposit in tissues and cause organ failure. Proteins form amyloid fibrils when they partly or fully unfold and expose segments capable of stacking into ß-sheets that pair and thereby form a tight, dehydrated interface. These structures, termed steric zippers, constitute the spines of amyloid fibrils. Here, using a combination of computational (with ZipperDB and Boston University ALBase), mutational, biochemical, and protein structural analyses, we identified segments within the variable domains of Ig light chains that drive the assembly of amyloid fibrils in AL. We demonstrate that there are at least two such segments and that each one can drive amyloid fibril assembly independently of the other. Our analysis revealed that peptides derived from these segments form steric zippers featuring a typical dry interface with high-surface complementarity and occupy the same spatial location of the Greek-key immunoglobulin fold in both λ and κ variable domains. Of note, some predicted steric-zipper segments did not form amyloid fibrils or assembled into fibrils only when removed from the whole protein. We conclude that steric-zipper propensity must be experimentally validated and that the two segments identified here may represent therapeutic targets. In addition to elucidating the molecular pathogenesis of AL, these findings also provide an experimental approach for identifying segments that drive fibril formation in other amyloid diseases.

Getting in charge of ß-synuclein fibrillation.

The synuclein family has long been associated with Parkinson's disease and dementia. Although the self-assembly of α-synuclein (αS) into oligomers and amyloid fibrils is well established, the aggregation propensity of other members of the family and their role in disease is still under debate. Moriarty et al. now suggest that the pH switching that occurs between different cellular environments could control ß-synuclein (ßS) aggregation via altering its charge distribution, thus opening new possible roles for ßS in Parkinson's and other neurodegenerative diseases.

The GPSM2/LGN GoLoco motifs are essential for hearing.

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn (ΔC) mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn (ΔC) allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.

Mutations in TAX1BP3 cause dilated cardiomyopathy with septo-optic dysplasia.

We describe a Bedouin family with a novel autosomal recessive syndrome characterized by dilated cardiomyopathy and septo-optic dysplasia. Genetic analysis revealed a homozygous missense mutation in TAX1BP3, which encodes a small PDZ domain containing protein implicated in regulation of the Wnt/ß-catenin signaling pathway, as the causative mutation. The mutation affects a conserved residue located at the core of TAX1BP3 binding pocket and is predicted to impair the nature of a crucial hydrophobic patch, thereby interrupting the structure and stability of the protein, and its ability to interact with other proteins. TAX1BP3 is highly expressed in heart and brain and consistent with the clinical findings observed in our patients; a knockdown of TAX1BP3 causes elongation defects, enlarged pericard, and enlarged head structures in zebrafish embryos. Thus, we describe a new genetic disorder that expands the monogenic cardiomyopathy disease spectrum and suggests that TAX1BP3 is essential for heart and brain development.

Formation of amyloid fibers by monomeric light chain variable domains.

Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers.

Towards a pharmacophore for amyloid.

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of ß-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases.

Molecular basis for amyloid-beta polymorphism.

Amyloid-beta (Aß) aggregates are the main constituent of senile plaques, the histological hallmark of Alzheimer's disease. Aß molecules form ß-sheet containing structures that assemble into a variety of polymorphic oligomers, protofibers, and fibers that exhibit a range of lifetimes and cellular toxicities. This polymorphic nature of Aß has frustrated its biophysical characterization, its structural determination, and our understanding of its pathological mechanism. To elucidate Aß polymorphism in atomic detail, we determined eight new microcrystal structures of fiber-forming segments of Aß. These structures, all of short, self-complementing pairs of ß-sheets termed steric zippers, reveal a variety of modes of self-association of Aß. Combining these atomic structures with previous NMR studies allows us to propose several fiber models, offering molecular models for some of the repertoire of polydisperse structures accessible to Aß. These structures and molecular models contribute fundamental information for understanding Aß polymorphic nature and pathogenesis.

Unique disulfide bonds in epidermal growth factor (EGF) domains of ß3 affect structure and function of αIIbß3 and αvß3 integrins in different manner.

The ß3 subunit of αIIbß3 and αvß3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbß3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbß3 and αvß3. We created ß3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbß3 and αvß3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbß3 and αvß3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbß3 and αvß3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbß3. Activation of C523S/C544S αvß3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S ß3 in αIIbß3 but not in αvß3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in ß3 differently affect the function of αIIbß3 and αvß3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbß3 and αvß3 and for Cys-523-Cys-544 only in αvß3.

Lipid bilayers significantly modulate cross-fibrillation of two distinct amyloidogenic peptides.

Amyloid plaques comprising misfolded proteins are the hallmark of several incurable diseases, including Alzheimer's disease, type-II diabetes, Jacob-Creutzfeld disease, and others. While the exact molecular mechanisms underlying protein misfolding diseases are still unknown, several theories account for amyloid fiber formation and their toxic significance. Prominent among those is the "prion hypothesis" stipulating that misfolded protein seeds act as "infectious agents" propagating aggregation of nominally healthy, native proteins. Recent studies, in fact, have reported that interactions between different amyloid peptides that are partly sequence-related might also affect fibrillation pathways and pathogenicity. Here, we present evidence that two structurally and physiologically unrelated amyloidogenic peptides, the islet amyloid polypeptide (IAPP, the peptide comprising the amyloid aggregates in type II diabetes) and an amyloidogenic determinant of the prion protein (PrP), give rise to a significantly distinct fibrillation pathway when they are incubated together in the presence of membrane bilayers. In particular, the experimental data demonstrate that the lipid bilayer environment is instrumental in initiating and promoting the assembly of morphologically distinct fibrillar species. Moreover, cross-fibrillation produced peptide species exhibiting significantly altered membrane interaction profiles, as compared to the scenario where the two peptides aggregated separately. Overall, our data demonstrate that membranes constitute a critical surface-active medium for promoting interactions between disparate amyloidogenic peptides, modulating both fibrillation pathways as well as the biophysical properties of the peptide aggregates. This work hints that membrane-induced cross-fibrillation of unrelated amyloidogenic peptides might play an insidious role in the molecular pathologies of protein misfolding diseases.

Compound heterozygosity of HLA-DRB3*01:01 and HLA-DRB4*01:01 as a potential predictor of fetal neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder in the fetus or neonate caused by maternal alloantibodies directed against fetal platelet (PLT) antigens inherited from the father. The immune-dominant antigen leading to severe FNAIT is the human PLT antigen (HPA)-1, whose polymorphism constitutes an epitope for human leukocyte antigens (HLAs), usually DRB3*0101 leading to an immune response. STUDY DESIGN AND METHODS: In this study our aims were to find whether other allele variants of the ß subunit of the HLA-DR family specifically focused on the HLA residues that bind Position 33 of the HPA-1 integrin contribute to FNAIT development and affect response to treatment and whether coexistence of both anti-HPA-1a and anti-HLA class I specific against the father's antigens leads to a more severe thrombocytopenia in the newborn. We examine the genotype of 23 mothers to newborns with FNAIT compared to a control group. RESULTS: Our results suggested that, when HPA-1 incompatibility with the husband is found, the presence of two HLA alleles (DRB3*01:01 and DRB4*01:01) in the mother increases the risk and severity of FNAIT and reduces the success of a preventive immunoglobulin G treatment. We provide a structural model for the molecular basis of the rational effects of the different HLA alleles. In addition, we found that the presence of both anti-HPA-1 and anti-HLAs did not aggravate FNAIT in comparison to mothers harboring only anti-HPA-1. CONCLUSION: Overall, we suggest that a specific genotyping of the mother in relation to HLA-DRB as well as HPA-1 can serve as an antenatal diagnostic tool, particularly in siblings of women who gave birth to neonates with FNAIT.

Beyond One-Trick Ponies: The Multifunctional Marvels of Microbial Functional Amyloids.

Various organisms, including bacteria, protists, fungi, plants, and animals, secrete proteins and peptides that self-assemble into ordered amyloid fibrils that perform different physiological functions [...].

Designed inhibitors to reduce amyloid virulence and cytotoxicity and combat neurodegenerative and infectious diseases.

The review highlights the role of amyloids in various diseases and the challenges associated with targeting human amyloids in therapeutic development. However, due to the better understanding of microbial amyloids' role as virulence factors, there is a growing interest in repurposing and designing anti-amyloid compounds for antivirulence therapy. The identification of amyloid inhibitors has not only significant clinical implications but also provides valuable insights into the structure and function of amyloids. The review showcases small molecules and peptides that specifically target amyloids in both humans and microbes, reducing cytotoxicity and biofilm formation, respectively. The review emphasizes the importance of further research on amyloid structures, mechanisms, and interactions across all life forms to yield new drug targets and improve the design of selective treatments. Overall, the review highlights the potential for amyloid inhibitors in therapeutic development for both human diseases and microbial infections.