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1.
Proc Natl Acad Sci U S A ; 117(11): 5844-5852, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32127471

RESUMO

Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (httex1) and responsible for Huntington's disease. Here, we investigate the interaction of profilin with httex1 using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in httex1, absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τex ≤ 50 to 70 µs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τex ∼750 µs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (K diss ∼ 17 and ∼ 31 µM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of httex1 with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale (τ ex ∼ 600 µs and K diss ∼ 50 µM). Finally, we demonstrate that, in stable profilin-httex1 complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil httex1 tetramers, is completely abolished, and only the pathway resulting in "nonproductive" dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of httex1.


Assuntos
Éxons , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Profilinas/química , Profilinas/metabolismo , Sítios de Ligação , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos , Conformação Proteica , Domínios Proteicos
2.
Mol Immunol ; 118: 210-221, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31901836

RESUMO

Eggplant or brinjal (Solanum melongena L.) is widely consumed worldwide and thought to trigger allergic reactions in sensitive individuals. So far, no molecular information is available on the allergy-eliciting components of eggplant. In this study, a 17 kDa profilin, Sola m 1, was identified from eggplant by employing an immunoproteomic approach. Based on MALDI-TOF/TOF derived sequences, the full-length cDNA of Sola m 1 was PCR amplified and then cloned. Recombinant (r) Sola m 1 was expressed in E. coli and then purified by metal affinity and gel filtration. rSola m 1 reacted with IgE-antibodies in the sera from all eggplant allergic patients. rSola m 1 also displayed allergenic activity by stimulating histamine release. rSola m 1 was monomeric, and the CD spectra revealed it to be folded with a mixture of α-helices and ß-strands. In the melting curve, rSola m 1 exhibited an irreversible denaturation where no refolding took place. Sola m 1 was found to share >80 % sequence identity with Bet v 2, which was further validated by confirming the presence of significant cross-reactivity with Bet v 2 in IgE-inhibition assay. IgE-cross reactivity was also observed between rSola m 1 and profilins from six other foods. In SGF assay, no rSola m 1-derived fragments exhibited IgE-reactivity after prolonged digestion suggesting the association of rSola m 1 with the oral allergy syndromes. Immunofluorescence localization revealed a high abundance of Sola m 1 allergen in eggplant seeds as compared to other edible parts. Taken together, Sola m 1 is the first major eggplant allergen reported in this study, which has the potential of being used as a candidate antigen in component-resolved diagnosis and immunotherapy.


Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Escherichia coli/genética , Profilinas/imunologia , Solanum melongena/imunologia , Adolescente , Adulto , Idoso , Reações Cruzadas/imunologia , DNA Complementar/genética , Feminino , Hipersensibilidade Alimentar/imunologia , Liberação de Histamina/imunologia , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Adulto Jovem
3.
J Cell Biol ; 219(1)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31816056

RESUMO

The development of cerebral cortex requires spatially and temporally orchestrated proliferation, migration, and differentiation of neural progenitor cells (NPCs). The molecular mechanisms underlying cortical development are, however, not fully understood. The neural cell adhesion molecule (NCAM) has been suggested to play a role in corticogenesis. Here we show that NCAM is dynamically expressed in the developing cortex. NCAM expression in NPCs is highest in the neurogenic period and declines during the gliogenic period. In mice bearing an NPC-specific NCAM deletion, proliferation of NPCs is reduced, and production of cortical neurons is delayed, while formation of cortical glia is advanced. Mechanistically, NCAM enhances actin polymerization in NPCs by interacting with actin-associated protein profilin2. NCAM-dependent regulation of NPCs is blocked by mutations in the profilin2 binding site. Thus, NCAM plays an essential role in NPC proliferation and fate decision during cortical development by regulating profilin2-dependent actin polymerization.


Assuntos
Antígeno CD56/fisiologia , Diferenciação Celular , Córtex Cerebral/citologia , Células-Tronco Neurais/citologia , Neurogênese , Neurônios/citologia , Profilinas/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Córtex Cerebral/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/metabolismo , Profilinas/genética
4.
Life Sci ; 254: 117180, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31863778

RESUMO

OBJECTIVE: Accumulating Studies implies that long-chain non-coding RNA (lncRNA) plays a vital regulatory role in the occurrence and progression of tumors. This study aimed to explore the function and mechanism of lncRNA HLA-F antisense RNA 1 (HLA-F-AS1) in colorectal cancer (CRC). METHODS: Expressions of HLA-F-AS1, miR-330-3p and profilin 1 (PFN1) mRNA in CRC tissues were detected by RT-PCR. MTT assay was used to detect cell proliferation, and Transwell assay was used to detect cell migration and invasion. In addition, PFN1 and apoptosis-related protein Bcl-2 associated X (Bax) and B cell lymphoma/leukmia-2 (Bcl2) were detected by western blot. Interactions between miR-330-3p and HLA-F-AS1 or the 3'UTR of PFN1 were predicted and determined by bioinformatics analysis and luciferase reporter assay. RESULTS: Expressions of HLA-F-AS1 and PFN1 were significantly up-regulated while miR-330-3p was significantly down-regulated in CRC tissues and cell lines. Over-expressions of HLA-F-AS1 or transfection of miR-330-3p inhibitors could promote the proliferation, migration and invasion and block apoptosis of CRC cells, whereas knockdown of HLA-F-AS1 or transfection of miR-330-3p mimics led to the opposite effects. Additionally, HLA-F-AS1 could down-regulate miR-330-3p via sponging it. HLA-F-AS1 also enhanced the expressions of PFN1, which was validated as a target gene of miR-330-3p. CONCLUSION: HLA-F-AS1 promoted CRC progression via regulating miR-330-3p/PFN1 axis.


Assuntos
Neoplasias Colorretais/patologia , Antígenos de Histocompatibilidade Classe I/metabolismo , MicroRNAs/metabolismo , Profilinas/metabolismo , Apoptose , Proliferação de Células , Neoplasias Colorretais/metabolismo , Progressão da Doença , Células HT29 , Humanos , Metástase Neoplásica
5.
Elife ; 82019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31647411

RESUMO

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.


Assuntos
Citoesqueleto de Actina/metabolismo , Forminas/metabolismo , Células Musculares/metabolismo , Profilinas/metabolismo , Multimerização Proteica , Animais , Mamíferos
6.
PLoS One ; 14(9): e0222697, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31545817

RESUMO

Dendritic cells (DCs) are professional antigen presenting cells involved in the induction of T cell-mediated adaptive immunity. Plasmacytoid DCs (pDCs) originate from lymphoid precursors and produce type I interferons (IFNs) in response to pathogens. A20 is considered as a negative regulator of toll-like receptor (TLR) signaling pathways, in which Toxoplasma gondii- derived profilin (TgPRF) is a TLR11/12 ligand recognised by DCs to stimulate their maturation/activation. Little is known about contributions of A20 to changes in biological properties of pDCs. The present study, therefore, explored whether pDC functions are influenced by A20. To this end, bone marrow cells were isolated and cultured with Flt3L to attain CD8DCs, CD11bDCs and pDCs and followed by challenge with TgPRP in the presence or absence of A20 siRNA. Expression of maturation markers were analysed by flow cytometry, and secretion of inflammatory cytokines by ELISA, cell migration by a transwell migration assay and expression of signalling molecules by western blotting. As a result, treatment with A20 siRNA enhanced activations of IκB-α and STAT-1, leading to increases in expressions of maturation markers and cytokine productions as well as migration of TgPRP-treated pDCs, while mature CD11bDCs produced at higher levels of TNF-α and IL-6 only. In addition, functions of CD8DCs remained unaltered following A20 silencing. The effects of A20 on pDC maturation and activation were completely abolished by IKK inhibitor and partially blunted by fludarabine. In conclusion, the inhibitory effects of A20 on pDC functions are expected to affect the immune response in T. gondii infection.


Assuntos
Células Dendríticas/fisiologia , NF-kappa B/fisiologia , Fator de Transcrição STAT1/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/fisiologia , Animais , Western Blotting , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Citometria de Fluxo , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Profilinas/farmacologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Toxoplasma/metabolismo
7.
Allergol Immunopathol (Madr) ; 47(6): 579-584, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31477404

RESUMO

BACKGROUND: In Portugal, the pollen types most implicated in respiratory allergy are grasses, olive and parietaria. The knowledge of sensitizations to molecular allergens in children and adults can contribute to better diagnosis and treatment of this pathology. METHODS: ImmunoCAP singleplex technology was used for molecular allergens and Phadia 250® automatic equipment. g205 (Phl p1); g215 (Phl p5b); g210 (Phl p7); and g212 (Phl p12) allergen determinations were made in 45 patients with positive grass sensitization tests. RESULTS: The majority of patients are sensitized to Phl p1 (91%) and Phl p1+/Phl p5-/Phl p7-/Phl p12- was the most dominant profile (40%). In the adult group, the IgE averages for Phl p1 were approximately 10.46, while they were 8.43 for Phl p5, 0.69 for Phl p7, and 0.06 for Phl p12. In the child group, these values were higher: 22.49, 20.23, 3.89, and 0.35, respectively. For allergens Phl p1, Phl p5, and Phl p7, these differences between the child and adult population were not statistically significant (p=0.754, p=0.806 and p=0.102, respectively), but for Phl p12, a statistically significant difference (p=0.018) was observed. CONCLUSIONS: IgE antibodies Phl p1 is the most important allergic marker and sensitivities caused by Phl p12 give rise to higher IgE values in children.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Imunização/estatística & dados numéricos , Proteínas de Plantas/imunologia , Profilinas/imunologia , Rinite Alérgica Sazonal/imunologia , Adolescente , Adulto , Idoso , Biomarcadores , Proteínas de Ligação ao Cálcio/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Imunoglobulina E/metabolismo , Masculino , Pessoa de Meia-Idade , Pólen/imunologia , Portugal/epidemiologia , Rinite Alérgica Sazonal/epidemiologia , Adulto Jovem
8.
Acta Crystallogr D Struct Biol ; 75(Pt 9): 852-860, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31478908

RESUMO

Theoretically, crystals with supercells exist at a unique crossroads where they can be considered as either a large unit cell with closely spaced reflections in reciprocal space or a higher dimensional superspace with a modulation that is commensurate with the supercell. In the latter case, the structure would be defined as an average structure with functions representing a modulation to determine the atomic location in 3D space. Here, a model protein structure and simulated diffraction data were used to investigate the possibility of solving a real incommensurately modulated protein crystal using a supercell approximation. In this way, the answer was known and the refinement method could be tested. Firstly, an average structure was solved by using the `main' reflections, which represent the subset of the reflections that belong to the subcell and in general are more intense than the `satellite' reflections. The average structure was then expanded to create a supercell and refined using all of the reflections. Surprisingly, the refined solution did not match the expected solution, even though the statistics were excellent. Interestingly, the corresponding superspace group had multiple 3D daughter supercell space groups as possibilities, and it was one of the alternate daughter space groups that the refinement locked in on. The lessons learned here will be applied to a real incommensurately modulated profilin-actin crystal that has the same superspace group.


Assuntos
Actinas/química , Cristalografia por Raios X/métodos , Profilinas/química , Modelos Moleculares , Conformação Proteica
9.
Nat Commun ; 10(1): 3827, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444357

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.


Assuntos
Actinas/metabolismo , Esclerose Amiotrófica Lateral/patologia , Neurônios Motores/patologia , Poro Nuclear/patologia , Profilinas/metabolismo , Acrilamidas/farmacologia , Actinas/ultraestrutura , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Esclerose Amiotrófica Lateral/genética , Biópsia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Embrião de Mamíferos , Fibroblastos , Humanos , Microscopia Eletrônica de Transmissão , Neurônios Motores/citologia , Mutação , Poro Nuclear/efeitos dos fármacos , Poro Nuclear/ultraestrutura , Cultura Primária de Células , Profilinas/genética , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Pele/citologia , Pele/patologia , Tiazóis/farmacologia , Tiazolidinas/farmacologia
10.
Pharmacol Rep ; 71(5): 940-949, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31454697

RESUMO

BACKGROUND: Furowanin A (Fur A) is a flavonoid compound isolated from medicinal plant Millettia pachycarpa Benth. This study aims to explore the effect of Fur A on Colorectal cancer (CRC) and its molecular mechanisms. METHODS: Cell proliferative capacity of CRC cells was assessed by CCK-8 assay. Cell apoptosis and cell cycle distribution were detected by flow cytometry. Cell migration and invasion were detected by wound healing and Transwell assay, respectively. EMT markers, apoptosis and profilin 1(Pfn1) expression were detected by immunohistochemistry (IHC). The protein expression levels were examined by western blotting. i-TRAQ analyses were conducted to identify the differentially expressed genes in CRC cells. CRC xenograft model was also used to validate the in vivo anti-cancer activity of Fur A. RESULTS: Fur A exhibited anti-prolifertive, blocked cell cycle progression and promoted apoptotic cell death in CRC cells. Fur A suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) in vitro, and tumor growth and pulmonary metastasis in vivo, without causing obvious toxicity. iTRAQ analysis identified Pfn1 as a gene up-regulated by Fur A. In xenograft tumor tissue, the expression of Pfn1 was also elevated by Fur A treatment. In clinical CRC samples, high expression of Pfn1 was correlated with lower stage and longer survival. Knockdown of Pfn1 significantly dampened the pro-apoptotic and anti-metastatic activities of Fur A in CRC cells. Ectopic Pfn1 expression augmented the anti-neoplastic activities of Fur A. CONCLUSION: Fur A exhibited anti-cancer activities in vitro and in vivo in CRC by up-regulating Pfn1.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Colorretais/metabolismo , Profilinas/metabolismo , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Millettia/química , Invasividade Neoplásica , Profilinas/genética , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cells ; 8(9)2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31450751

RESUMO

Skeletal muscle plays a crucial role in physical activity and in regulating body energy and protein balance. Myoblast proliferation, differentiation, and apoptosis are indispensable processes for myoblast myogenesis. Profilin 2a (PFN2a) is a ubiquitous actin monomer-binding protein and promotes lung cancer growth and metastasis through suppressing the nuclear localization of histone deacetylase 1 (HDAC1). However, how PFN2a regulates myoblast myogenic development is still not clear. We constructed a C2C12 mouse myoblast cell line overexpressing PFN2a. The CRISPR/Cas9 system was used to study the function of PFN2a in C2C12 myogenic development. We find that PFN2a suppresses proliferation and promotes apoptosis and consequentially downregulates C2C12 myogenic development. The suppression of PFN2a also decreases the amount of HDAC1 in the nucleus and increases the protein level of p53 during C2C12 myogenic development. Therefore, we propose that PFN2a suppresses C2C12 myogenic development via the p53 pathway. Si-p53 (siRNA-p53) reverses the PFN2a inhibitory effect on C2C12 proliferation and the PFN2a promotion effect on C2C12 apoptosis, and then attenuates the suppression of PFN2a on myogenic differentiation. Our results expand understanding of PFN2a regulatory mechanisms in myogenic development and suggest potential therapeutic targets for muscle atrophy-related diseases.


Assuntos
Apoptose , Profilinas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Camundongos , Desenvolvimento Muscular , Profilinas/genética
12.
Mol Immunol ; 114: 19-29, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326654

RESUMO

Worldwide, more than one-third of the population suffers from allergies. A significant fraction of officially registered allergens originate from the profilin family of proteins. Profilins are small ubiquitous proteins which are found in plants, viruses and various eukaryotes including mammals. Although they are primarily regarded as minor allergens, profilins are important players in immunoglobulin E (IgE) cross-reactivity. However, in some populations profilins are recognized by IgE from at least 50% of patients allergic to a given allergen source. Cuc m 2.0101 is recognized by IgE in more than 80% of muskmelon-allergic patients. The recombinant isoallergen Cuc m 2.0101 was produced in significant quantities and its X-ray crystal structure was determined. In addition, a new Art v 4.0101 (mugwort profilin) structure was determined. The profilins Cuc m 2.0101 and Art v 4.0101 were compared in terms of their structure and thermal stability. Furthermore, structural similarities and IgE cross-reactivity between profilins from different sources are discussed to explain the molecular basis of various clinical syndromes involving this group of allergens. Special emphasis is placed on discussion of profilins' quaternary structures and their relation to biological function, as well as to protein allergenicity. Moreover, a potential impact of protein purification protocols on the structure of profilins is highlighted.


Assuntos
Antígenos de Plantas/química , Profilinas/química , Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Reações Cruzadas/imunologia , Escherichia coli/imunologia , Escherichia coli/metabolismo , Hipersensibilidade/imunologia , Imunoglobulina E/química , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Pólen/química , Pólen/imunologia , Profilinas/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia
13.
Virology ; 533: 108-114, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31150988

RESUMO

We previously reported that human parainfluenza virus type 2 (hPIV-2) promoted RhoA activation and subsequent filamentous actin (F-actin) formation. Actin-binding proteins, such as profilin and cofilin, are involved in the regulation of F-actin formation by RhoA signaling. In the present study, we identified profilin2 as a key molecule that is involved in hPIV-2-induced F-actin formation. Immunoprecipitation assays demonstrated that hPIV-2 V protein binds to profilin2 but not to profilin1. Mutation of Trp residues within C-terminal region of V protein abolished the binding capacity to profilin2. Depletion of profilin2 resulted in the inhibition of hPIV-2-induced F-actin formation and the suppression of hPIV-2 growth. Overexpression of wild type V but not Trp-mutated V protein reduced the quantity of actin co-immunoprecipitated with profilin2. Taken together, these results suggest that hPIV-2 V protein promotes F-actin formation by affecting actin-profilin2 interaction through its binding to profilin2.


Assuntos
Actinas/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Profilinas/metabolismo , Infecções por Rubulavirus/metabolismo , Infecções por Rubulavirus/virologia , Actinas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Parainfluenza 2 Humana/genética , Profilinas/genética , Ligação Proteica , Infecções por Rubulavirus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Proc Natl Acad Sci U S A ; 116(26): 12629-12637, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31189606

RESUMO

The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how nonequilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Fatores de Despolimerização de Actina/farmacologia , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Forminas/metabolismo , Forminas/farmacologia , Profilinas/metabolismo , Profilinas/farmacologia
15.
PLoS One ; 14(6): e0215723, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31216283

RESUMO

Profilin 1 (PFN1) protein plays key roles in neuronal growth and differentiation, membrane trafficking, and regulation of the actin cytoskeleton. Four natural variants of PFN1 were described as related to ALS, the most common adult-onset motor neuron disorder. However, the pathological mechanism of PFN1 in ALS is not yet completely understood. The goal of this work is to thoroughly analyze the effects of the ALS-related mutations on PFN1 structure and function using computational simulations. Here, PhD-SNP, PMUT, PolyPhen-2, SIFT, SNAP, SNPS&GO, SAAP, nsSNPAnalyzer, SNPeffect4.0 and I-Mutant2.0 were used to predict the functional and stability effects of PFN1 mutations. ConSurf was used for the evolutionary conservation analysis, and GROMACS was used to perform the MD simulations. The mutations C71G, M114T, and G118V, but not E117G, were predicted as deleterious by most of the functional prediction algorithms that were used. The stability prediction indicated that the ALS-related mutations could destabilize PFN1. The ConSurf analysis indicated that the mutation C71G, M114T, E117G, and G118V occur in highly conserved positions. The MD results indicated that the studied mutations could affect the PFN1 flexibility at the actin and PLP-binding domains, and consequently, their intermolecular interactions. It may be therefore related to the functional impairment of PFN1 upon C71G, M114T, E117G and G118V mutations, and their involvement in ALS development. We also developed a database, SNPMOL (http://www.snpmol.org/), containing the results presented on this paper for biologists and clinicians to exploit PFN1 and its natural variants.


Assuntos
Esclerose Amiotrófica Lateral/genética , Polimorfismo de Nucleotídeo Único , Profilinas/química , Actinas/metabolismo , Sítios de Ligação , Sequência Conservada , Bases de Dados Genéticas , Humanos , Simulação de Dinâmica Molecular , Mutação , Profilinas/genética , Profilinas/metabolismo , Estabilidade Proteica
16.
PLoS Biol ; 17(6): e3000317, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181075

RESUMO

Within the cytoplasm of a single cell, several actin networks can coexist with distinct sizes, geometries, and protein compositions. These actin networks assemble in competition for a limited pool of proteins present in a common cellular environment. To predict how two distinct networks of actin filaments control this balance, the simultaneous assembly of actin-related protein 2/3 (Arp2/3)-branched networks and formin-linear networks of actin filaments around polystyrene microbeads was investigated with a range of actin accessory proteins (profilin, capping protein, actin-depolymerizing factor [ADF]/cofilin, and tropomyosin). Accessory proteins generally affected actin assembly rates for the distinct networks differently. These effects at the scale of individual actin networks were surprisingly not always correlated with corresponding loss-of-function phenotypes in cells. However, our observations agreed with a global interpretation, which compared relative actin assembly rates of individual actin networks. This work supports a general model in which the size of distinct actin networks is determined by their relative capacity to assemble in a common and competing environment.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/fisiologia , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Animais , Humanos , Cinética , Proteínas dos Microfilamentos/metabolismo , Profilinas/metabolismo , Mapas de Interação de Proteínas/fisiologia , Tropomiosina
17.
Medicina (Kaunas) ; 55(5)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117233

RESUMO

BACKGROUND AND OBJECTIVES: Nearly 20-30% of the world's population suffers from allergic rhinitis, among them 15% are progressing to asthma conditions. Sorghum bicolor profilin (Sorb PF), one of the panallergens, was identified, but the allergen specificity is not yet characterized. MATERIALS AND METHODS: To map the antigenic determinants responsible for IgE binding, the present study is focused on in silico modeling, simulation of Sorb PF and docking of the Sorb PF peptides (PF1-6) against IgG and IgE, followed by in vivo evaluation of the peptides for its allergenicity in mice. RESULTS: Peptide PF3 and PF4 displayed high docking G-scores (-9.05) against IgE only. The mice sensitized with PF3 peptide showed increased levels of IL5, IL12, TNF-alpha, and GMCSF when compared to other peptides and controls, signifying a strong, Th2-based response. Concurrently, the Th1 pathway was inhibited by low levels of cytokine IL2, IFN-γ, and IL-10 justifying the role of PF3 in allergenic IgE response. CONCLUSIONS: Based on the results of overlapping peptides PF3 and PF4, the N-terminal part of the PF3 peptide (TGQALVI) plays a crucial role in allergenic response of Sorghum profilin.


Assuntos
Simulação por Computador , Mapeamento de Peptídeos/métodos , Profilinas/análise , Sorghum/efeitos adversos , Animais , Modelos Animais de Doenças , Epitopos/análise , Camundongos , Profilinas/sangue , Sorghum/citologia
18.
Proc Natl Acad Sci U S A ; 116(24): 12084-12093, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31123151

RESUMO

Srv2p/CAP1 is an essential regulator of actin turnover, but its exact function in regulating actin polymerization, particularly the contribution of its actin nucleotide exchange activity, remains incompletely understood. We found that, although Arabidopsis CAP1 is distributed uniformly in the cytoplasm, its loss of function has differential effects on the actin cytoskeleton within different regions of the pollen tube. Specifically, the F-actin level increases in the shank but decreases in the apical region of cap1 pollen tubes. The reduction in apical F-actin results mainly from impaired polymerization of membrane-originated actin within cap1 pollen tubes. The actin nucleotide exchange activity of CAP1 is involved in apical actin polymerization. CAP1 acts synergistically with pollen ADF and profilin to promote actin turnover in vitro, and it can overcome the inhibitory effects of ADF and synergize with profilin to promote actin nucleotide exchange. Consistent with its role as a shuttle molecule between ADF and profilin, the cytosolic concentration of CAP1 is much lower than that of ADF and profilin in pollen. Thus, CAP1 synergizes with ADF and profilin to drive actin turnover in pollen and promote apical actin polymerization in pollen tubes in a manner that involves its actin nucleotide exchange activity.


Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Oligopeptídeos/metabolismo , Tubo Polínico/metabolismo , Citoesqueleto de Actina/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas dos Microfilamentos/metabolismo , Pólen/metabolismo , Polimerização , Profilinas/metabolismo
19.
Oncol Res ; 27(9): 1079-1088, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122311

RESUMO

Profilin 2 (PFN2) was found to be mainly expressed in neurons and involved in the development of the brain. In recent years, emerging evidence indicated that PFN2 is also significantly upregulated in various cancers including head and neck cancer (HNSC) and influences cancer cell proliferation, migration, and invasion. However, the role of PFN2 in HNSC development and progression remains unclear. The aim of our study was to investigate the role of PFN2 in the development of HNSC and its possible molecular mechanisms. Bioinformatics showed that increased expression of PFN2 in tumors correlated highly with poor prognosis of HNSC patients. Our results indicated that PFN2 was highly expressed in HNSC tissues and in HNSC cell lines. Knockdown of PFN2 inhibited proliferation, invasion, and migration of HNSC cells, while PFN2 overexpression produced the opposite effects. Using a nude mouse xenograft model, we substantiated the tumor-promoting effect of PFN2 on HNSC in vivo. Furthermore, we found that PFN2 downregulation reduced the phosphorylation of Akt and GSK-3ß and reduced the expression of ß-catenin in HNSC cells. The opposite was observed when PFN2 was overexpressed. Collectively, these results suggest that PFN2 promotes the proliferation and metastasis of HNSC by activating the PI3K/Akt/ß-catenin signaling pathway. Although further validation is needed, we speculate that PFN2 plays a crucial role in HNSC and may be a promising therapeutic target and prognostic biomarker.


Assuntos
Neoplasias de Cabeça e Pescoço/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Profilinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Xenoenxertos , Humanos , Camundongos , Metástase Neoplásica , Profilinas/biossíntese , Profilinas/genética , Transdução de Sinais , Regulação para Cima
20.
Comput Biol Chem ; 80: 177-186, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974345

RESUMO

Spatio-temporal expression patterns of cytoskeleton-associated profilin (PRF) family proteins in response to varied environmental stimuli are tightly regulated. Functional analyses of PRFs have revealed their crucial roles in varied developmental and stress related traits, but very little is implicit pertaining to cis-acting regulatory elements that regulate such intricate expression patterns. Here, we identified cis-elements with their varying distribution frequencies by scanning 1.5kbp upstream sequences of 5'regulatory regions of PRFs of dicot and monocot plant species. Predicted cis-elements in the regulatory sub-regions of Arabidopsis PRFs (AtPRFs) were predominantly associated with development-responsive motifs (DREs), light responsive elements (LREs), hormonal responsive elements (HREs), core motifs and stress-responsive elements (SREs). Interestingly, DREs, LREs and core promoter motifs, were extensively distributed up to the distal end of 5'regulatory regions on contrary to HREs present closer to the translational start site in Arabidopsis. The evolutionary footprints of predicted orthologous cis-elements were conserved, and preferably located in the proximal regions of 5'regulatory regions of evolutionarily diverged plant species. We also explored comprehensive tissue-specific global gene expression levels of PRFs under diverse hormonal and abiotic stress regimes. In response, the PRFs exhibited large transcriptional biases in a time- and organ-dependent manner. Further, the methodical elucidation of spatial expression analysis of predicted cis-elements binding transcription factors and relevant PRFs showed notable correlation. Results indicate that binding transcription factors' expression data is largely informative for envisaging their precise roles in the spatial regulation of target PRFs. These results highlight the importance of PRFs during plant development; and establish a relationship between their spatial expression patterns and presence of respective regulatory motifs in their promoter sequences. This information could be employed in future studies and field-utilization of cell wall structural genes.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Profilinas/genética , Elementos Reguladores de Transcrição , Sequência de Bases , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transcrição Genética
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