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.

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.

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.

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.

PFN4 is required for manchette development and acrosome biogenesis during mouse spermiogenesis.

Profilin 4 (Pfn4) is expressed during spermiogenesis and localizes to the acrosome-acroplaxome-manchette complex. Here, we generated PFN4-deficient mice, with sperm displaying severe impairment in manchette formation. Interestingly, HOOK1 staining suggests that the perinuclear ring is established; however, ARL3 staining is disrupted, suggesting that lack of PFN4 does not interfere with the formation of the perinuclear ring and initial localization of HOOK1, but impedes microtubular organization of the manchette. Furthermore, amorphous head shape and flagellar defects were detected, resulting in reduced sperm motility. Disrupted cis- and trans-Golgi networks and aberrant production of proacrosomal vesicles caused impaired acrosome biogenesis. Proteomic analysis showed that the proteins ARF3, SPECC1L and FKBP1, which are involved in Golgi membrane trafficking and PI3K/AKT pathway, are more abundant in Pfn4-/- testes. Levels of PI3K, AKT and mTOR were elevated, whereas AMPK level was reduced, consistent with inhibition of autophagy. This seems to result in blockage of autophagic flux, which could explain the failure in acrosome formation. In vitro fertilization demonstrated that PFN4-deficient sperm is capable of fertilizing zona-free oocytes, suggesting a potential treatment for PFN4-related human infertility.

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.

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.

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.

Yeast profilin mutants inhibit classical nuclear import and alter the balance between actin and tubulin levels.

Profilin is a small protein that controls actin polymerization in yeast and higher eukaryotes. In addition, profilin has emerged as a multifunctional protein that contributes to other processes in multicellular organisms. This study focuses on profilin (Pfy1) in the budding yeast Saccharomyces cerevisiae. The primary sequences of yeast Pfy1 and its metazoan orthologs diverge vastly. However, structural elements of profilin are conserved among different species. To date, the full spectrum of Pfy1 functions has yet to be defined. The current work explores the possible involvement of yeast profilin in nuclear protein import. To this end, a panel of well-characterized yeast profilin mutants was evaluated. The experiments demonstrate that yeast profilin (i) regulates nuclear protein import, (ii) determines the subcellular localization of essential nuclear transport factors, and (iii) controls the relative abundance of actin and tubulin. Together, these results define yeast profilin as a moonlighting protein that engages in multiple essential cellular activities.

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.

OptoProfilin: A Single Component Biosensor of Applied Cellular Stress.

The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as stress granules formed in response to energetic and oxidative stress are closely linked to ageing, cancer, cardiovascular disease, and viral infection. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin ('OptoProfilin') can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of preexisting work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.

Structural homology of mite profilins to plant profilins is not indicative of allergic cross-reactivity.

Structural and allergenic characterization of mite profilins has not been previously pursued to a similar extent as plant profilins. Here, we describe structures of profilins originating from Tyrophagus putrescentiae (registered allergen Tyr p 36.0101) and Dermatophagoides pteronyssinus (here termed Der p profilin), which are the first structures of profilins from Arachnida. Additionally, the thermal stabilities of mite and plant profilins are compared, suggesting that the high number of cysteine residues in mite profilins may play a role in their increased stability. We also examine the cross-reactivity of plant and mite profilins as well as investigate the relevance of these profilins in mite inhalant allergy. Despite their high structural similarity to other profilins, mite profilins have low sequence identity with plant and human profilins. Subsequently, these mite profilins most likely do not display cross-reactivity with plant profilins. At the same time the profilins have highly conserved poly(l-proline) and actin binding sites.

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.

Profilin promotes formin-mediated actin filament assembly and vesicle transport during polarity formation in pollen.

Pollen germination is critical for the reproduction of flowering plants. Formin-dependent actin polymerization plays vital roles in vesicle trafficking and polarity establishment during this process. However, how formin-mediated actin assembly is regulated in vivo remains poorly understood. Here, we investigated the function of reproductive profilin 4 and 5 (PRF4 and PRF5) in polarity establishment during pollen germination in Arabidopsis thaliana. Our data showed that the actin filament content was reduced in the prf4 prf5 double mutant and substantially increased in both PRF4- and PRF5-overexpressing pollen grains. By contrast, the positive effect of profilin in promoting actin polymerization was abolished in a formin mutant, atfh5. In addition, the interaction between Arabidopsis formin homology 5 (AtFH5) and actin filaments was attenuated and the trafficking of AtFH5-labeled vesicles was slowed in prf4 prf5 pollen grains. Formation of the collar-like structure at the germination pore was also defective in prf4 prf5 pollen grains as the fast assembly of actin filaments was impaired. Together, our results suggest that PRF4 and PRF5 regulate vesicle trafficking and polarity establishment during pollen germination by promoting AtFH5-mediated actin polymerization and enhancing the interaction between AtFH5 and actin filaments.

Genomes of Asgard archaea encode profilins that regulate actin.

The origin of the eukaryotic cell is unresolved1,2. Metagenomics sequencing has recently identified several potential eukaryotic gene homologues in Asgard archaea3,4, consistent with the hypothesis that the eukaryotic cell evolved from within the Archaea domain. However, many of these eukaryotic-like sequences are highly divergent and the organisms have yet to be imaged or cultivated, which brings into question the extent to which these archaeal proteins represent functional equivalents of their eukaryotic counterparts. Here we show that Asgard archaea encode functional profilins and thereby establish that this archaeal superphylum has a regulated actin cytoskeleton, one of the hallmarks of the eukaryotic cell5. Loki profilin-1, Loki profilin-2 and Odin profilin adopt the typical profilin fold and are able to interact with rabbit actin-an interaction that involves proteins from species that diverged more than 1.2 billion years ago6. Biochemical experiments reveal that mammalian actin polymerizes in the presence of Asgard profilins; however, Loki, Odin and Heimdall profilins impede pointed-end elongation. These archaeal profilins also retard the spontaneous nucleation of actin filaments, an effect that is reduced in the presence of phospholipids. Asgard profilins do not interact with polyproline motifs and the profilin-polyproline interaction therefore probably evolved later in the Eukarya lineage. These results suggest that Asgard archaea possess a primordial, polar, profilin-regulated actin system, which may be localized to membranes owing to the sensitivity of Asgard profilins to phospholipids. Because Asgard archaea are also predicted to encode potential eukaryotic-like genes involved in membrane-trafficking and endocytosis3,4, imaging is now necessary to elucidate whether these organisms are capable of generating eukaryotic-like membrane dynamics that are regulated by actin, such as are observed in eukaryotic cell movement, podosomes and endocytosis.

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

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.

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.

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.

Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex.

Capping protein (CP) is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between CP and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of CP and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility.