The advancements in proteomics have provided a better understanding of the functionality of apolipoproteins and lipoprotein-associated proteins, with the HDL lipoprotein fraction being the most studied. The focus of this study was to evaluate the HDL proteome in dyslipidemic subjects without an established cardiovascular disease, as well as to test whether rosuvastatin treatment alters the HDL proteome. Patients with primary hypercholesterolemia or mixed dyslipidemia were assigned to 20 mg/day rosuvastatin and blood samples were drawn at study entry and after 12 weeks of treatment. A label-free LC-MS/MS protein profiling was conducted, coupled with bioinformatics analysis. Sixty-nine HDL proteins were identified, belonging to four main biological function clusters: lipid transport and metabolism; platelet activation, degranulation, and aggregation, wound response and wound healing; immune response; inflammatory and acute phase response. Five HDL proteins showed statistically significant differences in the abundance (Anova ≤ 0.05), before and after rosuvastatin treatment. Platelet factor 4 variant (PF4V1), Pregnancy-specific beta-1-glycoprotein 2 (PSG2), Profilin-1 (PFN1) and Keratin type II cytoskeletal 2 epidermal (KRT2) showed decreased expressions, while Integrin alpha-IIb (ITGA2B) showed an increased expression after treatment with rosuvastatin. The ELISA validation of PFN1 segregated the subjects into responders and non-responders, as PFN1 levels after rosuvastatin were shown to mostly depend on the subjects\' inflammatory phenotype. Findings from this study introduce novel insights into the HDL proteome and statin pleiotropism.

Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

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.
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.
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.
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.

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, β-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 β-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.

Aggregation of profilin-1 (PFN1) causes a fatal neurodegenerative disease, familial amyotrophic lateral sclerosis (fALS). Histidine (His) tautomerism has been linked to the formation of fibril aggregation causing neurodegenerative disease. Characterization of intermediate species that form during aggregation is crucial, however, this has proven very challenging for experimentalists due to their transient nature. Hence, molecular dynamics (MD) simulations have been performed on the His tautomeric isomers εε, εδ, δε, and δδ of PFN1 to explain the structural changes and to correlate them with its aggregation propensity. MD simulations show that His133 presumably plays a major role in the aggregation of PFN1 upon His tautomerism compared to His119. Further, the formation of a new 310-helix is observed in εε and δε but 310-helix is not observed in δδ and εδ isomers. In addition, our findings unveil that β-sheet dominating conformations are observed in His119(δ)-His133(δ) δδ isomer of PFN1 with significant antiparallel β-sheets between residues T15-G23, S29-A33, L63-L65, Q68-S76, F83-T89, T97-T105, and K107-K115, suggesting a novel aggregation mechanism possibly occur for the formation of PFN1 aggregates. Overall, these results propose that MD simulations of PFN1 His tautomers can provide a detailed microscopic understanding of the aggregation mechanisms which are hard to probe through experiments.

BACKGROUND: Profilin-1 (Pfn1), an evolutionarily conserved actin-binding protein, is an important regulator of the cytoskeleton. We previously reported the osteoclast-specific Pfn1-conditional knockout (cKO) mice had postnatal osteolytic phenotype with craniofacial and long-bone deformities associated with increased migration of cultured osteoclasts. We hypothesized the increased cellular processes structured with branched actin filaments may underlies the mechanism of increased bone resorption in these mutant mice.
METHODS: The morphological structure and cell migration of the cultured osteoclasts were analyzed using fluorescent microscopy and time-lapse image capturing. Fractional migration distances, as well as the index of protrusive structures (%-PB) that evaluates relative border length of the protrusion were compared between the cells from control and Pfn1-cKO mice.
RESULTS: Time-lapse image analysis showed that %-PB was significantly larger in Pfn1-cKO osteoclasts. In addition, the fractional migration distance was positively correlated with the index. When the branched actin filament organization was suppressed by chemical inhibitors, the osteoclast migration was declined. Importantly, the suppression was more extensive in Pfn1-cKO than in control osteoclasts.
CONCLUSIONS: Our results indicated the causative involvement of the increased branched actin filament formation at least in part for their excessive migration. Our findings provide a mechanistic rationale for testing novel therapeutic approaches targeting branched actin filaments in osteolytic disorders.

BACKGROUND: The accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic vasculopathy. However, the role of profilin-1 as a multifunctional actin-binding protein in AGEs-induced atherosclerosis (AS) is largely unknown.
OBJECTIVE: To explore the potential role of profilin-1 in the pathogenesis of AS induced by AGEs, particularly in relation to the Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signaling pathway.
METHODS: Eighty-nine individuals undergoing coronary angiography were enrolled in the study. Plasma cytokine levels were detected using ELISA kits. Rat aortic vascular smooth muscle cells (RASMCs) were incubated with different compounds for different times. Cell proliferation was determined by performing the MTT assay and EdU staining. An AGEs-induced vascular remodeling model was established in rats and histological and immunohistochemical analyses were performed. The mRNA and protein levels were detected using real-time PCR and Western blot analysis, respectively. In vivo, shRNA transfection was performed to verify the role of profilin-1 in AGEs-induced proatherogenic mediator release and aortic remodeling. Statistical analyses were performed using SPSS 22.0 software.
RESULTS: Compared with the control group, plasma levels of profilin-1 and receptor for AGEs (RAGE) were significantly increased in patients with coronary artery disease, especially in those complicated with diabetes mellitus (P < 0.01). The levels of profilin-1 were positively correlated with the levels of RAGE (P < 0.01); additionally, the levels of both molecules were positively associated with the degree of coronary artery stenosis (P < 0.01). In vivo, tail vein injections of AGEs induced the release of proatherogenic mediators, such as asymmetric dimethylarginine, intercellular adhesion molecule-1, and the N-terminus of procollagen III peptide, concomitant with apparent aortic morphological changes and significantly upregulated expression of the profilin-1 mRNA and protein in the thoracic aorta (P < 0.05 or P < 0.01). Downregulation of profilin-1 expression with an shRNA significantly attenuated AGEs-induced proatherogenic mediator release (P < 0.05) and aortic remodeling. In vitro, incubation of vascular smooth muscle cells (VSMCs) with AGEs significantly promoted cell proliferation and upregulated the expression of the profilin-1 mRNA and protein (P < 0.05). AGEs (200 μg/mL, 24 h) significantly upregulated the expression of the STAT3 mRNA and protein and JAK2 protein, which was blocked by a JAK2 inhibitor (T3042-1) and/or STAT3 inhibitor (T6308-1) (P < 0.05). In addition, pretreatment with T3042-1 or T6308-1 significantly inhibited AGEs-induced RASMC proliferation (P < 0.05).
CONCLUSIONS: AGEs induce proatherogenic events such as VSMC proliferation, proatherogenic mediator release, and vascular remodeling, changes that can be attenuated by silencing profilin-1 expression. These results suggest a crucial role for profilin-1 in AGEs-induced vasculopathy.

Previous studies have demonstrated that the levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, are strongly associated with hypertension, diabetes, and cardiovascular diseases. Profilin-1, an actin-binding protein, has been documented to be involved in endothelial injury and in the proliferation of vascular smooth muscle cells resulting from hypertension. However, the role of profilin-1 in ADMA-induced vascular injury in hypertension remains largely unknown. Forty healthy subjects and forty-two matched patients with essential hypertension were enrolled, and the related indexes of vascular injury in plasma were detected. Rat aortic smooth muscle cells (RASMCs) were treated with different concentrations of ADMA for different periods of time and transfected with profilin-1 small hairpin RNA to interrupt the expression of profilin-1. To determine the role of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway, RASMCs were pretreated with AG490 or rapamycin. The expression of profilin-1 was tested using real-time polymerase chain reaction (PCR) and western blot analysis. Cell proliferation was measured by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide assays. Compared with healthy subjects, the levels of ADMA and profilin-1 were markedly elevated in hypertensive individuals, while the levels of NO were significantly decreased (p < 0.05). In vitro, studies showed ADMA-induced profilin-1 expression in a concentration- and time-dependent manner in RASMCs (p < 0.05), concomitantly with promoting the proliferation of RASMCs. Furthermore, ADMA-mediated proliferation of RASMCs and upregulation expression of profilin-1 were inhibited by blockade of the JAK2/STAT3 pathway or knockdown of profilin-1. Profilin-1 implicated in the ADMA-mediated vascular lesions in hypertension.

The essential actin-binding factor profilin-1 (Pfn1) is a non-classical tumor suppressor with the abilities toboth inhibit cellular proliferation and augment chemotherapy-induced apoptosis. Besides actin, Pfn1 interacts with proteins harboring the poly-L-proline (PLP) motifs. Our recent work demonstrated that both nuclear localization and PLP-binding are required for tumor growth inhibition by Pfn1, and this is at least partially due to Pfn1 association with the PLP-containing ENL protein in the Super Elongation Complex (SEC) and the transcriptional inhibition of pro-cancer genes. In this paper, by identifying a phosphorylation event of Pfn1 at Ser71 capable of inhibiting its actin-binding and nuclear export, we provide in vitro and in vivo evidence that chemotherapy-induced apoptotic sensitization by Pfn1 requires its cytoplasmic localization and actin-binding. With regard to tumor growth inhibition byPfn1, our data indicate a requirement for dynamic actin association and dissociation rendered by reversible Ser71phosphorylation and dephosphorylation. Furthermore, genetic and pharmacological experiments showed that Ser71 of Pfn1 can be phosphorylated by protein kinase A (PKA). Taken together, our data provide novel mechanistic insights into the multifaceted anticancer activities of Pfn1 and how they are spatially-defined in the cell and differentially regulated by ligand-binding.

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.