Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.

Orosomucoid 2 as a biomarker of carotid artery atherosclerosis plaque vulnerability through its generation of reactive oxygen species and lipid accumulation in vascular smooth muscle cells.

BACKGROUND: Orosomucoid (ORM) has been reported as a biomarker of carotid atherosclerosis, but the role of ORM 2, a subtype of ORM, in carotid atherosclerotic plaque formation and the underlying mechanism have not been established. METHODS: Plasma was collected from patients with carotid artery stenosis (CAS) and healthy participants and assessed using mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) technology to identify differentially expressed proteins. The key proteins and related pathways were identified via western blotting, immunohistochemistry, and polymerase chain reaction of carotid artery plaque tissues and in vitro experiments involving vascular smooth muscle cells (VSMCs). RESULTS: We screened 33 differentially expressed proteins out of 535 proteins in the plasma. Seventeen proteins showed increased expressions in the CAS groups relative to the healthy groups, while 16 proteins showed decreased expressions during iTRAQ and bioinformatic analysis. The reactive oxygen species metabolic process was the most common enrichment pathway identified by Gene Ontology analysis, while ORM2, PRDX2, GPX3, HP, HBB, ANXA5, PFN1, CFL1, and S100A11 were key proteins identified by STRING and MCODE analysis. ORM2 showed increased expression in patients with CAS plaques, and ORM2 was accumulated in smooth muscle cells. Oleic acid increased the lipid accumulation and ORM2 and PRDX6 expressions in the VSMCs. The recombinant-ORM2 also increased the lipid accumulation and reactive oxygen species (ROS) in the VSMCs. The expressions of ORM2 and PRDX-6 were correlated, and MJ33 (an inhibitor of PRDX6-PLA2) decreased ROS production and lipid accumulation in VSMCs. CONCLUSION: ORM2 may be a biomarker for CAS; it induced lipid accumulation and ROS production in VSMCs during atherosclerosis plaque formation. However, the relationships between ORM2 and PRDX-6 underlying lipid accumulation-induced plaque vulnerability require further research.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control.

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

Mechanisms of actin disassembly and turnover.

Cellular actin networks exhibit a wide range of sizes, shapes, and architectures tailored to their biological roles. Once assembled, these filamentous networks are either maintained in a state of polarized turnover or induced to undergo net disassembly. Further, the rates at which the networks are turned over and/or dismantled can vary greatly, from seconds to minutes to hours or even days. Here, we review the molecular machinery and mechanisms employed in cells to drive the disassembly and turnover of actin networks. In particular, we highlight recent discoveries showing that specific combinations of conserved actin disassembly-promoting proteins (cofilin, GMF, twinfilin, Srv2/CAP, coronin, AIP1, capping protein, and profilin) work in concert to debranch, sever, cap, and depolymerize actin filaments, and to recharge actin monomers for new rounds of assembly.

Long noncoding RNA RMRP ameliorates doxorubicin-induced apoptosis by interacting with PFN1 in a P53-Dependent manner.

Doxorubicin (DOX) often causes acute or chronic cardiotoxicity during its application. LncRNA RMRP has been reported to be associated with several biological processes, such as cartilage-hair hypoplasia, but the relationship between RMRP and DOX-induced cardiotoxicity and chronic heart failure remains obscure. To test this hypothesis, GSE124401 and GSE149870 were processed for bioinformatics, and differentially expressed RMRP was then verified in the peripheral blood of 21 patients with heart failure compared with 7 controls. For in vitro validation, we used AC16 and HEK-293T cells. qPCR was used to detect the mRNA expression levels. The degree of apoptosis was detected by Western blot and TUNEL staining. Furthermore, the interaction between RMRP and PFN1 mRNA was verified by dual-luciferase reporter assays. In bioinformatics, RMRP showed significant downregulation, which was verified in clinical samples (p < 0.001) and DOX-treated AC16 models (p < 0.0001). Next, overexpression of RMRP could significantly alleviate DOX-induced apoptosis, and a potential downstream molecule of RMRP, PFN1, was also negatively associated with this change. RESCUE experiments further confirmed that PFN1 could be regulated by RMRP at both the RNA and protein levels, serving as a downstream mediator of RMRP's cardioprotective effects. This interaction was then confirmed to be a direct combination (p < 0.0001). Finally, we found that overexpression of RMRP could inhibit the expression of p53 and its phosphorylation level by suppressing PFN1. In summary, RMRP could exert cardioprotective effects via the PFN1/p53 axis, holding great promise for serving as a therapeutic target and potential biomarker.

Cyclase-associated protein interacts with actin filament barbed ends to promote depolymerization and formin displacement.

Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments. In the absence of actin monomers, full-length mouse CAP1 and C-terminal halves of CAP1 (C-CAP1) and CAP2 (C-CAP2) accelerate barbed end depolymerization. Using mutagenesis and structural modeling, we show that these activities are mediated by the WH2 and CARP domains of CAP. In addition, we observe that CAP collaborates with profilin to accelerate barbed end depolymerization and that these effects depend on their direct interaction, providing the first known example of CAP-profilin collaborative effects in regulating actin. In the presence of actin monomers, CAP1 attenuates barbed end growth and promotes formin dissociation. Overall, these findings demonstrate that CAP uses distinct domains and mechanisms to interact with opposite ends of actin filaments and drive turnover. Further, they contribute to the emerging view of actin barbed ends as sites of dynamic molecular regulation, where numerous proteins compete and cooperate with each other to tune polymer dynamics, similar to the rich complexity seen at microtubule ends.

Neutrophil attachment via Mac-1 (αMß2; CD11b/CD18; CR3) integrins induces PAD4 deimination of profilin and histone H3.

Neutrophil adhesion to endothelia, entry into tissues and chemotaxis constitute essential steps in the immune response to infections that drive inflammation. Neutrophils bind to other cells and migrate via adhesion receptors, notably the αMß2 integrin dimer (also called Mac-1, CR3 or CD11b/CD18). Here, the response of neutrophils to integrin engagement was examined by monitoring the activity of peptidylarginine deiminase 4 (PAD4). Histone H3 deimination was strongly stimulated by manganese, an integrin-activating divalent cation, even in the absence of additional inflammatory stimuli. Manganese-induced cell attachment resulted in neutrophil swarm formation that paralleled histone deimination, whereas antibodies that impair integrin binding prevented both cell adhesion and histone deimination. Manganese treatment led to putative deimination of profilin, a protein that functions as an actin-organizing hub, as detected by two-dimensional gel electrophoresis and citrulline immunoblotting. Cl-amidine, a covalent inhibitor of PAD4, and GSK484, a specific PAD4 inhibitor, blocked profilin deimination. Neutrophil migration toward leukotriene B4 and toward synovial fluid from a rheumatoid arthritis patient were inhibited by chloramidine, thus supporting the contribution of deimination to chemotaxis. The data, based on a simplified system for integrin activation, imply a mechanism whereby integrin attachment coordinates neutrophil responses to inflammation and orchestrates deimination of nuclear and cytoskeletal proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Proximity Proteomics Revealed Aberrant mRNA Splicing Elicited by ALS-Linked Profilin-1 Mutants.

Profilin 1 (PFN1) is a cytoskeleton protein that modulates actin dynamics through binding to monomeric actin and polyproline-containing proteins. Mutations in PFN1 have been linked to the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Here, we employed an unbiased proximity labeling strategy in combination with proteomic analysis for proteome-wide profiling of proteins that differentially interact with mutant and wild-type (WT) PFN1 proteins in human cells. We uncovered 11 mRNA splicing proteins that are preferentially enriched in the proximity proteomes of the two ALS-linked PFN1 variants, C71G and M114T, over that of wild-type PFN1. We validated the preferential interactions of the ALS-linked PFN1 variants with two mRNA splicing factors, hnRNPC and U2AF2, by immunoprecipitation, followed with immunoblotting. We also found that the two ALS-linked PFN1 variants promoted the exonization of Alu elements in the mRNAs of MTO1, TCFL5, WRN and POLE genes in human cells. Together, we showed that the two ALS-linked PFN1 variants interacted preferentially with mRNA splicing proteins, which elicited aberrant exonization of the Alu elements in mRNAs. Thus, our work provided pivotal insights into the perturbations of ALS-linked PFN1 variants in RNA biology and their potential contributions to ALS pathology.

Vascular endothelial profilin-1 drives a protumorigenic tumor microenvironment and tumor progression in renal cancer.

Overexpression of actin-binding protein profilin-1 (Pfn1) correlates with advanced disease features and adverse clinical outcome of patients with clear cell renal carcinoma, the most prevalent form of renal cancer. We previously reported that Pfn1 is predominantly overexpressed in tumor-associated vascular endothelial cells in human clear cell renal carcinoma. In this study, we combined in vivo strategies involving endothelial cell-specific depletion and overexpression of Pfn1 to demonstrate a role of vascular endothelial Pfn1 in promoting tumorigenicity and enabling progressive growth and metastasis of renal carcinoma cells in a syngeneic orthotopic mouse model of kidney cancer. We established an important role of endothelial Pfn1 in tumor angiogenesis and further identified endothelial Pfn1-dependent regulation of several pro- (VEGF, SERPINE1, CCL2) and anti-angiogenic factors (platelet factor 4) in vivo. Endothelial Pfn1 overexpression increases tumor infiltration by macrophages and concomitantly diminishes tumor infiltration by T cells including CD8+ T cells in vivo, correlating with the pattern of endothelial Pfn1-dependent changes in tumor abundance of several prominent immunomodulatory cytokines. These data were also corroborated by multiplexed quantitative immunohistochemistry and immune deconvolution analyses of RNA-seq data of clinical samples. Guided by Upstream Regulator Analysis of tumor transcriptome data, we further established endothelial Pfn1-induced Hif1α elevation and suppression of STAT1 activation. In conclusion, this study demonstrates for the first time a direct causal relationship between vascular endothelial Pfn1 dysregulation, immunosuppressive tumor microenvironment, and disease progression with mechanistic insights in kidney cancer. Our study also provides a conceptual basis for targeting Pfn1 for therapeutic benefit in kidney cancer.

Profilin1 Regulates Trophoblast Invasion and Macrophage Differentiation.

Unexplained recurrent spontaneous abortion (URSA) has been associated with the dysfunction of trophoblasts and decidual macrophages. Current evidence suggests that profilin1 (PFN1) plays an important role in many biological processes. However, little is known about whether PFN1 is related to URSA. Herein, the location of PFN1 was detected by immunohistochemistry, and the level of PFN1 was detected by quantitative real-time PCR, Western blot analysis, and immunohistochemistry. The proliferation of trophoblasts was detected by CCK8 and 5-ethynyl-2'-deoxyuridine assays, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were used to detect apoptosis of trophoblasts. The migration and invasion ability of trophoblasts was assessed by using the wound-healing test and transwell test. Polarization of macrophages was detected in macrophages cultured in trophoblast conditioned medium. PFN1 expression was observed in cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts and was decreased in the villous tissue of patients with URSA. The migration and invasion ability and cell viability of trophoblastic cell lines that underwent PFN1 knockdown significantly decreased, and apoptosis increased. Opposite findings were observed after the overexpression of PFN1 in trophoblastic cells. In addition, PFN1 could regulate trophoblast function through phosphatidylinositol 3-kinase/AKT signal transduction rather than mitogen-activated protein kinase signaling pathways. Finally, knockdown of PFN1 in trophoblasts promoted tumor necrosis factor-α secretion to induce macrophage polarization to M1 phenotype, mediated by the NF-κB signaling pathway. These findings indicate that PFN1 has a broad therapeutic potential for patients with URSA.

Molecular Basis of Plant Profilins' Cross-Reactivity.

Profilins are ubiquitous allergens with conserved structural elements. Exposure to profilins from different sources leads to IgE-cross-reactivity and the pollen-latex-food syndrome. Monoclonal antibodies (mAbs) that cross-react with plant profilins and block IgE-profilin interactions are relevant for diagnosis, epitope mapping, and specific immunotherapy. We generated IgGs mAbs, 1B4, and 2D10, against latex profilin (anti-rHev b 8) that inhibit the interaction of IgE and IgG4 antibodies from sera of latex- and maize-allergic patients by 90% and 40%, respectively. In this study, we evaluated 1B4 and 2D10 recognition towards different plant profilins, and mAbs recognition of rZea m 12 mutants by ELISAs. Interestingly, 2D10 highly recognized rArt v 4.0101 and rAmb a 8.0101, and to a lesser extent rBet v 2.0101, and rFra e 2.2, while 1B4 showed recognition for rPhl p 12.0101 and rAmb a 8.0101. We demonstrated that residue D130 at the α-helix 3 in profilins, which is part of the Hev b 8 IgE epitope, is essential for the 2D10 recognition. The structural analysis suggests that the profilins containing E130 (rPhl p 12.0101, rFra e 2.2, and rZea m 12.0105) show less binding with 2D10. The distribution of negative charges on the profilins' surfaces at the α-helices 1 and 3 is relevant for the 2D10 recognition, and that may be relevant to explain profilins' IgE cross-reactivity.

Purification of human ß- and γ-actin from budding yeast.

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human ß- or γ-actin (i.e. cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both ß- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin and thymosin-ß4 (Tß4). Notably, Tß4 and profilin bind to ß- or γ-actin with higher affinity than to α-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies on actin regulation.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Role of profilin-1 in vasculopathy induced by advanced glycation end products (AGEs).

AIMS: To construct a simple and feasible rat model to mimic diabetic vasculopathy by chronic injection of advanced glycation end products (AGEs) and further determine the role of profilin-1 in vasculopathy in AGE-injection rats. METHODS: Sprague-Dawley rats were injected with AGEs-BSA (25 mg/kg/day) for 0, 20, 30, 40, and 60 days by caudal vein. Then, the morphological changes in the aorta, heart, and kidney and the expression of profilin-1 were assessed. In cultured endothelial cells, shRNA profilin-1 was used to clarify the role of profilin-1 in AGEs-induced vascular endothelial lesions and inflammatory reactions. RESULTS: The aorta, heart, and kidney of the AGE-injection rats had obvious morphological changes. Also, the indicators of vascular remodeling in the aorta significantly increased, accompanied by the increased expression of profilin-1 in the aorta, heart, and kidney and polysaccharide content on the kidney basement membrane. In addition, the protein level of profilin-1 was markedly upregulated in the aorta of AGEs-injected rats and endothelial cells incubated with AGEs. shRNA profilin-1 markedly attenuated the upregulated expression of profilin-1, receptor for AGEs (RAGE), and NF-κB in endothelial cells incubated with AGEs, as well as reduced the high levels of ICAM-1, IL-8, TNF-α, ROS, and apoptosis induced by AGEs. CONCLUSIONS: Exogenous AGEs can mimic diabetic vasculopathy in vivo to some extent and increase profilin-1 expression in the target organs of diabetic complications. Blockade of profilin-1 attenuates vascular lesions and inflammatory reactions, suggesting its critical role in the metabolic memory mediated by AGEs.

Upregulation of Profilin 2 on HDAC6 overexpression in mouse GC-1 cells and its putative role in germ cell migration in the testis.

Previous reports from this laboratory have demonstrated the involvement of histone deacetylase 6 (HDAC6) in sperm motility. As the presence of HDAC6 has also been reported in the earlier stage germ cells, studies were undertaken to explore its role during these stages of spermatogenesis. HDAC6 was overexpressed in GC-1spg cells, which represent the stage between type B spermatogonia and primary spermatocyte, and its effect on germ cell transcriptome was investigated by microarray. Among the many transcripts that were differentially regulated, Profilin 2, reported previously as a neuronal specific isoform, was observed as one of the genes highly upregulated at the transcript level, which was further confirmed by real-time PCR, and the protein confirmed by indirect immunofluorescence (IIF). Profilin 2 colocalized with HDAC6, as seen both in GC-1 cells and sperm. On the sperm, the presence of Profilin 2 was detected throughout the flagella with its colocalization with HDAC6 seen conspicuously in the mid-piece region of the flagella. Co-immunoprecipitation studies confirmed Profilin 2 interaction with HDAC6. Docking studies using Z dock suggested the interaction of 8 residues of HDAC6 with 6 residues of Profilin 2. The novel observation of Profilin 2 in spermatogonial cells, its significant upregulation on HDAC6 overexpression and its interaction with HDAC6 suggests that HDAC6 in collaboration with Profilin 2 may play a role in regulating the movement of germ cells from one stage/compartment to the next.

Profilin 1 deficiency drives mitotic defects and reduces genome stability.

Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability.

Mechanistic insights into the conformational switch in profilin-1 subject to collective effects of mutation and histidine tautomerism.

Mutations and histidine (His) tautomerism in profilin-1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS). The conformational changes in PFN1 caused by the collective effects of G117V mutation and His tautomeric isomers εε, εδ, δε, and δδ were clarified using molecular dynamics (MD) simulations. The predominant structural variations were seen in α-helices, ß-sheets, turns, and coils and the His tautomer's unique degree of disruption was seen in these conformations. The content of α-helices was 23.2 % in the εε and δδ isomers, but the observed α-helices in the isomers εδ and δε were 20.3 % and 21.7 % respectively. The percentage of ß-sheet was found to be higher (34.1) in the εε isomer than in the εδ, δε, and δδ isomers, and the values were 30.4, 29.7, and 31.9, respectively. Intermolecular water dynamics analysis discloses that His 133 can form an intramolecular H-bond interaction (Nα-H---Nδ), confirming the experimental observations in the simulations of εε, δε, and δδ isomers of G117V PFN1 mutant. It was concluded that these solvent molecules are crucial for aggregation and must be considered in future research on the PFN1 associated with ALS. Overall, the study offers a thorough microscopic understanding of the pathogenic mechanisms behind conformational changes that cause aggregation illnesses like ALS.

AlphaFold2: A versatile tool to predict the appearance of functional adaptations in evolution: Profilin interactions in uncultured Asgard archaea: Profilin interactions in uncultured Asgard archaea.

The release of AlphaFold2 (AF2), a deep-learning-aided, open-source protein structure prediction program, from DeepMind, opened a new era of molecular biology. The astonishing improvement in the accuracy of the structure predictions provides the opportunity to characterize protein systems from uncultured Asgard archaea, key organisms in evolutionary biology. Despite the accumulation in metagenomics-derived Asgard archaea eukaryotic-like protein sequences, limited structural and biochemical information have restricted the insight in their potential functions. In this review, we focus on profilin, an actin-dynamics regulating protein, which in eukaryotes, modulates actin polymerization through (1) direct actin interaction, (2) polyproline binding, and (3) phospholipid binding. We assess AF2-predicted profilin structures in their potential abilities to participate in these activities. We demonstrate that AF2 is a powerful new tool for understanding the emergence of biological functional traits in evolution.

Effects of covalent conjugation with quercetin and its glycosides on the structure and allergenicity of Bra c p from bee pollen.

Profilin family members are potential pan-allergens in foods, presenting public health hazards. However, studies on the allergenicity modification of profilin allergens are limited. Herein, quercetin and its glycosides (isoquercitrin and rutin) were applied to modify the allergenicity of a profilin allergen (Bra c p) from Brassica campestris bee pollen. Results showed that only quercetin can be closely covalently bound to Bra c p among the three, and the binding site was located at the Cys98 residue. After covalently conjunction, the relative content of α-helix structure in Bra c p was reduced by 40.05%, while random coil was increased by 42.89%; moreover, the Tyr and Phe residues in Bra c p were masked. These structural changes could alter the conformational antigenic epitopes of Bra c p, resulting in its allergenicity reduction. Our findings might provide a technical foundation for reducing the allergenicity of bee pollen and foods containing profilin family allergens.