Haplo-insufficiency of Profilin1 in vascular endothelial cells is beneficial but not sufficient to confer protection against experimentally induced atherosclerosis.

Actin cytoskeleton plays an important role in various aspects of atherosclerosis, a key driver of ischemic heart disease. Actin-binding protein Profilin1 (Pfn1) is overexpressed in atherosclerotic plaques in human disease, and Pfn1, when partially depleted globally in all cell types, confers atheroprotection in vivo. This study investigates the impact of endothelial cell (EC)-specific partial loss of Pfn1 expression in atherosclerosis development. We utilized mice engineered for conditional heterozygous knockout of the Pfn1 gene in ECs, with atherosclerosis induced by depletion of hepatic LDL receptor by gene delivery of PCSK9 combined with high-cholesterol diet. Our studies show that partial depletion of EC Pfn1 has certain beneficial effects marked by dampening of select pro-atherogenic cytokines (CXCL10 and IL7) with concomitant reduction in cytotoxic T cell abundance but is not sufficient to reduce hyperlipidemia and confer atheroprotection in vivo. In light of these findings, we conclude that atheroprotective phenotype conferred by global Pfn1 haplo-insufficiency requires contributions of additional cell types that are relevant for atherosclerosis progression.

Swimming alters some proteins of skeletal muscle tissue in rats with Alzheimer-like phenotype.

OBJECTIVES: Exercise training plays a significant role in preventing the destruction of central nerve neurons and muscle atrophy. The purpose of the present study was to investigate the effect of a period of swimming training on the expression of Neural cell adhesion molecule (NCAM), Semaphorin 3A (SEMA3A), and Profilin-1 (PFN1) proteins in the gastrocnemius muscle of Alzheimer-like phenotype rats. METHODS & MATERIALS: 32 Wistar males were (6 weeks of age) divided into four groups: Healthy Control (HC), Alzheimer-like phenotype's Control (AC), Healthy Training (HT), and Alzheimer-like phenotype's Training (AT). Alzheimer-like phenotypes were induced by beta-amyloid injection in the hippocampus. The training program consisted of 20 swimming sessions. Gastrocnemius muscle was removed after the intervention, and NCAM, SEMA3A, and PFN1 proteins were measured by the immunohistoflorescent method. RESULTS: The results showed that SEMA3A was increased (p = 0.001), and NCAM (p = 0.001), and PFN1 (p = 0.001) were decreased in AC compared to the HC group. Also, the results showed that NCAM (p = 0.001) and Pfn1 (p = 0.002) increased in the HT group compared to HC, and the NCAM (p = 0.001) and Pfn1 (p = 0.002) in AT group compared to AC (p = 0.001) increased significantly, while SEMA3A was reduced in the HT group compared to HC (p = 0.001) and AT group compared to AC (p = 0.001) CONCLUSION: Swimming effectively improves axon regeneration and neuronal formation in motor neurons and, therefore, can be an effective intervention to prevent and control the complications of Alzheimer-like phenotype.

Osteolytic Bone Loss and Skeletal Deformities in a Mouse Model for Early-Onset Paget's Disease of Bone with PFN1 Mutation Are Treatable by Alendronate.

A novel osteolytic disorder due to PFN1 mutation was discovered recently as early-onset Paget's disease of bone (PDB). Bone loss and pain in adult PDB patients have been treated using bisphosphonates. However, therapeutic strategies for this specific disorder have not been established. Here, we evaluated the efficiency of alendronate (ALN) on a mutant mouse line, recapitulating this disorder. Five-week-old conditional osteoclast-specific Pfn1-deficient mice (Pfn1-cKOOCL) and control littermates (33 females and 22 males) were injected with ALN (0.1 mg/kg) or vehicle twice weekly until 8 weeks of age. After euthanizing, bone histomorphometric parameters and skeletal deformities were analyzed using 3D µCT images and histological sections. Three weeks of ALN administration significantly improved bone mass at the distal femur, L3 vertebra, and nose in Pfn1-cKOOCL mice. Histologically increased osteoclasts with expanded distribution in the distal femur were normalized in these mice. Geometric bone shape analysis revealed a partial recovery from the distal femur deformity. A therapeutic dose of ALN from 5 to 8 weeks of age significantly improved systemic bone loss in Pfn1-cKOOCL mice and femoral bone deformity. Our study suggests that preventive treatment of bony deformity in early-onset PDB is feasible.

Rosuvastatin effects on the HDL proteome in hyperlipidemic patients.

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.

Genetic Screening of ZNF687 and PFN1 in a Paget's Disease of Bone Cohort Indicates an Important Role for the Nuclear Localization Signal of ZNF687.

Paget's disease of bone (PDB) is a common, late-onset bone disorder, characterized by focal increases of bone turnover that can result in bone lesions. Heterozygous pathogenic variants in the Sequestosome 1 (SQSTM1) gene are found to be the main genetic cause of PDB. More recently, PFN1 and ZNF687 have been identified as causal genes in patients with a severe, early-onset, polyostotic form of PDB, and an increased likelihood to develop giant cell tumors. In our study, we screened the coding regions of PFN1 and ZNF687 in a Belgian PDB cohort (n = 188). In the PFN1 gene, no variants could be identified, supporting the observation that variants in this gene are extremely rare in PDB. However, we identified 3 non-synonymous coding variants in ZNF687. Interestingly, two of these rare variants (p.Pro937His and p.Arg939Cys) were clustering in the nuclear localization signal of the encoded ZNF687 protein, also harboring the p.Pro937Arg variant, a previously reported disease-causing variant. In conclusion, our findings support the involvement of genetic variation in ZNF687 in the pathogenesis of classical PDB, thereby expanding its mutational spectrum.

The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence.

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.

ALS-causing hPFN1 mutants differentially disrupt LLPS of FUS prion-like domain.

hPFN1 mutations including C71G cause ALS by gain of toxicity but the mechanism still remains unknown. Stress granules (SGs) are formed by phase separation of the prion-like domain (PLD) of RNA-binding proteins including FUS, whose inclusion was also associated with ALS. C71G-hPFN1 triggers seed-dependent co-aggregation with FUS/TDP-43 to manifest the prion-like propagandation but its biophysical basis remains unexplored. Here by DIC imaging we first showed that three hPFN1 mutants have differential capacity in disrupting the dynamics of liquid droplets formed by phase separation of FUS prion-like domain (PLD). C71G-hPFN1 co-exists with the folded and unfolded states, thus allowing to simultaneously characterize conformations, hydrodynamics and dynamics of the interactions of both states with the phase separated FUS PLD by NMR. The results reveal that the folded state is not significantly affected while by contrast, the unfolded state has extensive interactions with FUS PLD. As a consequence, the dynamics of FUS liquid droplets become significantly reduced. Such interactions might act to recruit C71G-hPFN1 into the droplets, thus leading to the increase of the local concentrations and subsequent co-aggregation of C71G-hPFN1 with FUS. Our study sheds the first light on the biophysical basis by which hPFN1 mutants gain toxicity to cause ALS. As other aggregation-prone proteins have no fundamental difference from hPFN1 mutants, aggregation-prone proteins might share a common capacity in disrupting phase separation responsible for organizing various membrane-less organelles. As such, the mechanism for C71G-hPFN1 might also be utilized by other aggregation-prone proteins for gain of toxicity to trigger diseases and aging.

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.

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.

BAL Proteomic Signature of Lung Adenocarcinoma in IPF Patients and Its Transposition in Serum Samples for Less Invasive Diagnostic Procedures.

Idiopathic pulmonary fibrosis (IPF) is a form of chronic and irreversible fibrosing interstitial pneumonia of unknown etiology. Although antifibrotic treatments have shown a reduction of lung function decline and a slow disease progression, IPF is characterize by a very high mortality. Emerging evidence suggests that IPF increases the risk of lung carcinogenesis. Both diseases show similarities in terms of risk factors, such as history of smoking, concomitant emphysema, and viral infections, besides sharing similar pathogenic pathways. Lung cancer (LC) diagnosis is often difficult in IPF patients because of the diffuse lung injuries and abnormalities due to the underlying fibrosis. This is reflected in the lack of optimal therapeutic strategies for patients with both diseases. For this purpose, we performed a proteomic study on bronchoalveolar lavage fluid (BALF) samples from IPF, LC associated with IPF (LC-IPF) patients, and healthy controls (CTRL). Molecular pathways involved in inflammation, immune response, lipid metabolism, and cell adhesion were found for the dysregulated proteins in LC-IPF, such as TTHY, APOA1, S10A9, RET4, GDIR1, and PROF1. The correlation test revealed a relationship between inflammation- and lipid metabolism-related proteins. PROF1 and S10A9, related to inflammation, were up-regulated in LC-IPF BAL and serum, while APOA1 and APOE linked to lipid metabolism, were highly abundant in IPF BAL and low abundant in IPF serum. Given the properties of cytokine/adipokine of the nicotinamide phosphoribosyltransferase, we also evaluated its serum abundance, highlighting its down-regulation in LC-IPF. Our retrospective analyses of BAL samples extrapolated some potential biomarkers of LC-IPF useful to improve the management of these contemporary pathologies. Their differential abundance in serum samples permits the measurement of these potential biomarkers with a less invasive procedure.

Histidine tautomerism dependent conformational transitions driven aggregation of profilin-1: Implications in amyotrophic lateral sclerosis.

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 ß-sheet dominating conformations are observed in His119(δ)-His133(δ) δδ isomer of PFN1 with significant antiparallel ß-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.

Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer.

Profilin 1 (PFN1), an actin-binding protein, plays contrasting roles in the metastasis of several cancers; however, its role in non-small cell lung cancer (NSCLC) metastasis remains unclear. Here, PFN1 expression was upregulated in metastatic NSCLC tissues. PFN1 overexpression significantly promotes NSCLC metastasis in vitro and in vivo. Proteomics analysis revealed PFN1 involvment in microvesicles (MVs) secretion. In vitro experiments confirmed that PFN1 overexpression increased secretion of MVs. MVs are important mediators of metastasis. Here, we show an increased abundance of MVs in the sera of patients with metastatic NSCLC compared to that in the sera of patients with non-metastatic NSCLC. Both in vitro and in vivo experiments revealed that PFN1 could increase MV secretion, and MVs derived from PFN1-overexpressing cells markedly promoted NSCLC metastasis. We then elucidated the mechanisms underlying PFN1-mediated regulation of MVs and found that PFN1 could interact with ROCK1 and enhance its kinase activity to promote myosin light chain (MLC) phosphorylation for MV secretion. Inhibition of ROCK1 decreased MV secretion and partially reversed the PFN1-induced promotion of NSCLC metastasis. Collectively, these findings show that PFN1 regulates MV secretion to promote NSCLC metastasis. PFN1 and MVs represent potential predictors or therapeutic targets for NSCLC metastasis.

Profilin-1 negatively controls osteoclast migration by suppressing the protrusive structures based on branched actin filaments.

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. MATERIALS AND 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. CONCLUSION: 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.

Effect of profilin-1 on the asymmetric dimethylarginine-induced vascular lesion-associated hypertension.

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.

Profilin-1 is involved in macroangiopathy induced by advanced glycation end products via vascular remodeling and inflammation.

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. AIM: 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). CONCLUSION: 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.

Upregulated Expression of Profilin1 on Dendritic Cells in Patients With Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is a life-threatening form of bone marrow failure that is associated with very high mortality. Dendritic cells (DCs) are antigen presenting cells (APCs) with powerful movement ability, which is an important factor affecting immune function. The expression of profilin1 (Pfn1) plays an important role in the regulation of cell movement ability. We detected the expression of Pfn1 mRNA in the bone marrow (BM) myeloid dendritic cells (mDCs) from patients with SAA using RT-PCR. Next, we examined Pfn1 expression on mDCs using flow cytometry (FCM). We also assessed the relationship between Pfn1 expression and cytokine levels. Our data showed increased Pfn1 mRNA expression in patients with SAA. The expression of Pfn1 in BM mDCs increased in SAA patients. The expression of Pfn1 on mDCs and cytokines (TNF-α and IFN-γ) were positively correlated in the serum of untreated patients with SAA. Taken together, we found that the expression of Pfn1 on mDCs of SAA patients increased, which may affect the function of mDCs. Profilin 1 may be involved in the immunopathogenesis of SAA.

Ser71 Phosphorylation Inhibits Actin-Binding of Profilin-1 and Its Apoptosis-Sensitizing Activity.

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.

Prognostic and clinicopathologic value of ki-67 and profilin 1 immunohistochemical expression in primary pT1 urothelial bladder cancer.

PURPOSE: To investigate the prognostic and clinicopathologic value of Ki-67 and profilin 1 immunohistochemical expression in primary pT1 papillary urothelial bladder cancer. MATERIALS AND METHODS: This study included 88 male and 13 female pT1 primary bladder cancer patients. Demographic characteristics, tumor histological grade, tumor number, presence of concomitant carcinoma in situ, tumor size, and status of recurrence or progression were recorded for each patient. Expression of Ki-67 and profilin 1 was evaluated by immunohistochemical analysis of paraffin-embedded tumor tissues. The Pearson's Chi-square test was used for the analysis of qualitative data, and the Kaplan-Meier method and the log-rank test were used for the survival analysis. RESULTS: In the mean follow-up period of 52 months, 52 (51.5%) patients experienced recurrence, 24 (23.8%) patients experienced progression, and 17 (16.8%) patients died from bladder cancer-related causes. Ki-67 expression was significantly associated with tumor histological grade (P = 0.001). In multivariate analysis, Ki-67 positivity had significantly worse outcome for recurrence (P = 0.006) and mortality (P = 0.022). Ki-67-positive (Ki-67 index ≥15%) patients had shorter recurrence-free (P = 0.003), progression-free (P = 0.002), and cancer-specific (P = 0.003) survival. However, no statistically significant relationship was found between profilin 1 expression and clinicopathologic features and prognosis. CONCLUSIONS: Ki-67 is a highly predictive biomarker for recurrence-free, progression-free, and cancer-specific survival in pT1 bladder cancer patients, in whom prediction of recurrence and progression are difficult. Ki-67 expression can be safely combined with other prognostic factors. However, in pT1 bladder cancer patients, no significant relationship was found between profilin 1 expression and tumor characteristics or prognostic parameters.

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.

Profilin-1; a novel regulator of DNA damage response and repair machinery in keratinocytes.

Profilin-1 (PFN1) regulates actin polymerization and cytoskeletal growth. Despite the essential roles of PFN1 in cell integration, its subcellular function in keratinocyte has not been elucidated yet. Here we characterize the specific regulation of PFN1 in DNA damage response and repair machinery. PFN1 depletion accelerated DNA damage-mediated apoptosis exhibiting PTEN loss of function instigated by increased phosphorylated inactivation followed by high levels of AKT activation. PFN1 changed its predominant cytoplasmic localization to the nucleus upon DNA damage and subsequently restored the cytoplasmic compartment during the recovery time. Even though γH2AX was recruited at the sites of DNA double strand breaks in response to DNA damage, PFN1-deficient cells failed to recruit DNA repair factors, whereas control cells exhibited significant increases of these genes. Additionally, PFN1 depletion resulted in disruption of PTEN-AKT cascade upon DNA damage and CHK1-mediated cell cycle arrest was not recovered even after the recovery time exhibiting γH2AX accumulation. This might suggest PFN1 roles in regulating DNA damage response and repair machinery to protect cells from DNA damage. Future studies addressing the crosstalk and regulation of PTEN-related DNA damage sensing and repair pathway choice by PFN1 may further aid to identify new mechanistic insights for various DNA repair disorders.