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.

Innovative electrochemical platform for the simultaneous determination of L-DOPA and L-tyrosine using layer-by-layer assembled L-proline-linked nanodiamonds on printed graphene.

Discovering alternative analytical techniques is crucial for practical applications; thus, this work aims to develop an innovative and simple electrochemical sensor for melanoma and the clinical diagnosis of related disorders by the simultaneous determination of 3,4-dihydroxy-L-phenylalanine (L-DOPA) and L-tyrosine (L-Tyr). The fabrication is based on the layer-by-layer electrodeposition of poly L-proline (poly(L-pro)) and nanodiamond (ND) onto a screen-printed graphene electrode (SPGE). The poly(L-pro)/ND/SPGEs were morphologically characterized by scanning electron microscopy, energy-dispersive X-ray spectrometry, and Raman spectroscopy followed by electrochemical investigation using cyclic voltammetry, differential pulse voltammetry, chronoamperometry, and electrochemical impedance spectroscopy. These modifier-based electrodes pave a feasible way to unlock the coexisting interfering substances from screen-printing ink composition and improve the sensitivity. Additionally, computational chemistry calculations were performed to fully comprehend the sensing behavior on both target analytes. Under optimal conditions, the developed sensor provided linear concentration ranges of 0.075-50 µM, with a detection limit of 0.021 µM for L-DOPA, and 2.5-120 µM with a detection limit of 0.74 µM for L-Tyr. To demonstrate the reliability of the poly(L-pro)/ND/SPGE in practical application, it was successfully applied to the determination of these analytes in human urine and blood serum samples, with satisfactory recovery ranges (81.73-110.62% for L-DOPA and 82.17-110.01% for L-Tyr) and relative standard deviations (0.69-9.90% for L-DOPA and 0.40-9.55% for L-Tyr). Due to its simplicity, long-term stability (> 87.8% of their initial currents after 35 days), and portability, the developed sensor is a promising alternative analytical method for on-site clinical monitoring.

Simultaneous Electrochemical Sensing of Indole-3-Acetic Acid and Salicylic Acid on Poly(L-Proline) Nanoparticles-Carbon Dots-Multiwalled Carbon Nanotubes Composite-Modified Electrode.

Sensitive simultaneous electrochemical sensing of phytohormones indole-3-acetic acid and salicylic acid based on a novel poly(L-Proline) nanoparticles-carbon dots composite consisting of multiwalled carbon nanotubes was reported in this study. The poly(L-Proline) nanoparticles-carbon dots composite was facilely prepared by the hydrothermal method, and L-Proline was used as a monomer and carbon source for the preparation of poly(L-Proline) nanoparticles and carbon dots, respectively. Then, the poly(L-Proline) nanoparticles-carbon dots-multiwalled carbon nanotubes composite was prepared by ultrasonic mixing of poly(L-Proline) nanoparticles-carbon dots composite dispersion and multiwalled carbon nanotubes. Scanning electron microscope, transmission electron microscope, Fourier transform infrared spectroscopy, ultraviolet visible spectroscopy, energy dispersive spectroscopy, cyclic voltammetry, electrochemical impedance spectroscopy, and linear sweep voltammetry were used to characterize the properties of the composite. poly(L-Proline) nanoparticles were found to significantly enhance the conductivity and sensing performance of the composite. Under optimal conditions, the composite-modified electrode exhibited a wide linear range from 0.05 to 25 µM for indole-3-acetic acid and from 0.2 to 60 µM for salicylic acid with detection limits of 0.007 µM and 0.1 µM (S/N = 3), respectively. In addition, the proposed sensor was also applied to simultaneously test indole-3-acetic acid and salicylic acid in real leaf samples with satisfactory recovery.

Profilin2a-phosphorylation as a regulatory mechanism for actin dynamics.

Profilin is a major regulator of actin dynamics in multiple specific processes localized in different cellular compartments. This specificity is not only meditated by its binding to actin but also its interaction with phospholipids such as phosphatidylinositol (4,5)-bisphosphate (PIP2 ) at the membrane and a plethora of proteins containing poly-L-proline (PLP) stretches. These interactions are fine-tuned by posttranslational modifications such as phosphorylation. Several phospho-sites have already been identified for profilin1, the ubiquitously expressed isoform. However, little is known about the phosphorylation of profilin2a. Profilin2a is a neuronal isoform important for synapse function. Here, we identified several putative profilin2a phospho-sites in silico and tested recombinant phospho-mimetics with regard to their actin-, PLP-, and PIP2 -binding properties. Moreover, we assessed their impact on actin dynamics employing a pyrene-actin polymerization assay. Results indicate that distinct phospho-sites modulate specific profilin2a functions. We could identify a molecular switch site at serine residue 71 which completely abrogated actin binding-as well as other sites important for fine-tuning of different functions, for example, tyrosine 29 for PLP binding. Our findings suggest that differential profilin2a phosphorylation is a sensitive mechanism for regulating its neuronal functions. Moreover, the dysregulation of profilin2a phosphorylation may contribute to neurodegeneration.

Monolithic silica columns functionalized with substituted polyproline-derived chiral selectors as chiral stationary phases for high-performance liquid chromatography.

In this study, two polyproline-derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead-based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.

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.

Actin sequestering protein, profilin, regulates intracellular vesicle transport in Leishmania.

Profilins are the key regulators of actin dynamics in all eukaryotic cells. However, little information is available on their biochemical properties and functions in kinetoplastids, such as Trypanosoma and Leishmania. We show here that Leishmania parasites express only one homolog of profilin (LdPfn), which catalyzes nucleotide exchange on G-actin and promotes actin polymerization at its low concentrations. However, at high concentrations, it strongly inhibits the polymerization process by sequestering actin monomers. We further demonstrate that LdPfn binds to actin in Leishmania promastigotes, by both immunofluorescence microscopy and IgG affinity chromatography. Further, we reveal that this protein besides binding to poly-null-proline motifs, also binds more efficiently to PI(3,5)P2, which is found on early or late endosomes or lysosomes, than to PI(4,5)P2 and PI(3,4,5)P3. Additionally, we show that heterozygous mutants of profilin display significantly slower growth and intracellular vesicle trafficking activity, which is reversed on episomal gene complementation. Together, these findings suggest that profilin regulates intracellular vesicle trafficking in Leishmania perhaps through its binding to polyphosphoinositides.

Monolithic silica columns with covalently attached octaproline chiral selector. Dependence of performance on derivatization degree and comparison with a bead-based analogue.

A monolithic silica gel chromatographic matrix was derivatized repetitively with an octaproline-derived chiral selector (CS). The increasingly derivatized column was tested after each derivatization reaction. The enantioseparation ability, resolution and efficiency were found to depend on the content of CS attained after each reaction. Moreover, enantioselectivity and performance of the column with the highest CS coverage were compared to those of a bead-based chiral stationary phase (CSP) counterpart. The octaproline-derivatized monolithic column demonstrated increased enantioseparation factors, resolution and broader applicability than the particle-based column. Finally, the loading capacity of the CSPs was also examined. The monolithic octaproline-derived column permits the separation of 3-20 times higher molar amounts of the tested analytes (depending on the compound considered) than the particle-based counterpart. The enhanced capabilities of the derivatized monolithic column with respect to that of a bead-based counterpart cannot be explained only on the basis of an increased CS coverage. The involvement of an effect produced by the chromatographic silica support structure in the obtained results is discussed.

Effects of supercritical fluid chromatography conditions on enantioselectivity and performance of polyproline-derived chiral stationary phases.

The chromatographic behaviour and performance of four polyproline-derived chiral stationary phases (CSPs) were tested using supercritical fluid chromatography (SFC). A series of structurally related racemic compounds, whose enantioseparation was proved to be sensitive to the type of mobile phase used in NP-HPLC, were chosen to be tested in the SFC conditions. Good enantioselection ability was shown by the CSPs for the analytes tested in the new conditions. Resolution, efficiency and analysis time, were considerably improved with respect to NP-HPLC when CO2/alcohol mobile phases were used. Monolithic columns clearly show enhanced chromatographic parameters and improved performance respect to their bead-based counterparts.

Octaproline, a conformationally flexible chiral selector in liquid chromatographic enantioseparation.

A proline octapeptide-derived chiral selector (CS) end-capped using a pivaloyl group was covalently linked to a silica gel chromatographic matrix by the C-terminal group. The chromatographic behaviour of the resulting chiral stationary phase (CSP) using different conditions was compared to those containing 3,5-dimethylphenylcarbamate residues on the proline units. An enantioseparation ability highly dependent on the mobile phase used is observed for these CSPs. When mixtures of alkane/alcohol or alkane/ether are used as mobile phase a similar enantioselectivity is obtained. Nevertheless, in the presence of chlorinated solvents, and without a hydrogen bonding donor in the mobile phase, enantioselectivity is extremely reduced. The reversibility of this phenomenon, attributed to a conformational change in the CS, is examined.

Structure and functions of profilins.

Profilins are small actin-binding proteins found in eukaryotes and certain viruses that are involved in cell development, cytokinesis, membrane trafficking, and cell motility. Originally identified as an actin sequestering/binding protein, profilin has been involved in actin polymerization dynamics. It catalyzes the exchange of ADP/ATP in actin and increases the rate of polymerization. Profilins also interact with polyphosphoinositides (PPI) and proline-rich domains containing proteins. Through its interaction with PPIs, profilin has been linked to signaling pathways between the cell membrane and the cytoskeleton, while its role in membrane trafficking has been associated with its interaction with proline-rich domain-containing proteins. Depending on the organism, profilin is present in a various number of isoforms. Four isoforms of profilin have been reported in higher organisms, while only one or two isoforms are expressed in single-cell organisms. The affinity of these isoforms for their ligands varies between isoforms and should therefore modulate their functions. However, the significance and the functions of the different isoforms are not yet fully understood. The structures of many profilin isoforms have been solved both in the presence and the absence of actin and poly-L-proline. These structural studies will greatly improve our understanding of the differences and similarities between the different profilins. Structural stability studies of different profilins are also shedding some light on our understanding of the profilin/ligand interactions. Profilin is a multifaceted protein for which a dramatic increase in potential functions has been found in recent years; as such, it has been implicated in a variety of physiological and pathological processes.