The amino acid sequence of Acanthamoeba profilin

The complete amino acid sequence of Acanthamoeba profilin was determined by aligning tryptic, chymotryptic, thermolysin, and Staphylococcus aureus V8 protease peptides together with the partial NH2-terminal sequences of the tryptophan-cleavage products. Acanthamoeba profilin contains 125 amino acid residues, is NH2-terminally blocked, and has trimethyllysine at position 103. At five positions in the sequence two amino acids were identified indicating that the amoebae express at least two slightly different profilins. Charged residues are unevenly distributed, the NH2-terminal half being very hydrophobic and the COOH-terminal half being especially rich in basic residues. Comparison of the Acanthamoeba profilin sequence with that of calf spleen profilin (Nystrom, L. E., Lindberg, U., Kendrick-Jones, J., and Jakes, R. (1979) FEBS Lett. 101, 161-165) reveals homology in the NH2-terminal region. We suggest, therefore, that this region participates in the actin-binding activity.     

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.     

Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells

We have previously isolated and crystallized a complex from calf spleen, containing actin and a smaller protein which we call profilin. In this paper we describe some properties of this complex, and show that association with profilin is sufficient to explain the persistent monomeric state of some of the actin in spleen extracts; moreover, spleen profilin will recombine with skeletal muscle actin to form a non-polymerizable complex resembling that isolated from spleen. Profilin is not restricted to spleen, but is found in a variety of other tissues and tissue-cultured cell lines. We propose that reversible association of actin with profilin in the cell may provide a mechanism for storage of monomeric actin and controlled turnover of microfilaments.     

High-resolution structural analysis of mammalian profilin 2a complex formation with two physiological ligands: the formin homology 1 domain of mDia1 and the proline-rich domain of VASP

Profilins are small proteins capable of binding actin, poly-l-proline and other proline-rich sequences, and phosphatidylinositol (4,5)-bisphosphate. A number of proline-rich ligands for profilin have been characterised, including proteins of the Ena/VASP and formin families. We have determined the high-resolution crystal structures of mouse profilin 2a in complex with peptides from two functionally important ligands from different families, VASP and mDia1. The structures show that the binding mode of the peptide ligand is strongly affected by the non-proline residues in the sequence, and the peptides from VASP and mDia1 bind to profilin 2a in distinct modes. The high resolution of the crystallographic data allowed us to detect conserved CH-π hydrogen bonds between the peptide and profilin in both complexes. Furthermore, both peptides, which are shown to have micromolar affinity, induced the dimerisation of profilin, potentially leading to functionally different ligand-profilin-actin complexes. The peptides did not significantly affect actin polymerisation kinetics in the presence or in the absence of profilin 2a. Mutant profilins were tested for binding to poly-l-proline and the VASP and mDia1 peptides, and the F139A mutant bound proline-rich ligands with near-native affinity. Peptide blotting using a series of designed peptides with profilins 1 and 2a indicates differences between the two profilins towards proline-rich peptides from mDia1 and VASP. Our data provide structural insights into the mechanisms of mDia1 and VASP regulated actin polymerisation.     

Refined solution structure of human profilin I.

Profilin is a ubiquitous eukaryotic protein that binds to both cytosolic actin and the phospholipid phosphatidylinositol-4,5-bisphosphate. These dual competitive binding capabilities of profilin suggest that profilin serves as a link between the phosphatidyl inositol cycle and actin polymerization, and thus profilin may be an essential component in the signaling pathway leading to cytoskeletal rearrangement. The refined three-dimensional solution structure of human profilin I has been determined using multidimensional heteronuclear NMR spectroscopy. Twenty structures were selected to represent the solution conformational ensemble. This ensemble of structures has root-mean-square distance deviations from the mean structure of 0.58 A for the backbone atoms and 0.98 A for all non-hydrogen atoms. Comparison of the solution structure of human profilin to the crystal structure of bovine profilin reveals that, although profilin adopts essentially identical conformations in both states, the solution structure is more compact than the crystal structure. Interestingly, the regions that show the most structural diversity are located at or near the actin-binding site of profilin. We suggest that structural differences are reflective of dynamical properties of profilin that facilitate favorable interactions with actin. The global folding pattern of human profilin also closely resembles that of Acanthamoeba profilin I, reflective of the 22% sequence identity and approximately 45% sequence similarity between these two proteins.     

The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.     

Phosphorylation of profilin by ROCK1 regulates polyglutamine aggregation

Y-27632, an inhibitor of the Rho-associated kinase ROCK, is a therapeutic lead for Huntington disease (HD). The downstream targets that mediate its inhibitory effects on huntingtin (Htt) aggregation and toxicity are unknown. We have identified profilin, a small actin-binding factor that also interacts with Htt, as being a direct target of the ROCK1 isoform. The overexpression of profilin reduces the aggregation of polyglutamine-expanded Htt and androgen receptor (AR) peptides. This requires profilin's G-actin binding activity and its direct interaction with Htt, which are both inhibited by the ROCK1-mediated phosphorylation of profilin at Ser-137. Y-27632 blocks the phosphorylation of profilin in HEK293 cells and primary neurons, which maintains profilin in an active state. The knockdown of profilin blocks the inhibitory effect of Y-27632 on both AR and Htt aggregation. A signaling pathway from ROCK1 to profilin thus controls polyglutamine protein aggregation and is targeted by a promising therapeutic lead for HD.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Specificity of the interaction between phosphatidylinositol 4,5-bisphosphate and the profilin:actin complex

Profilactin, the profilin:actin complex, which is present in large amounts in extracts of many types of eukaryotic cells, appears to serve as the precursor of microfilaments. It was reported recently that profilactin interacts specifically with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (Lassing and Lindberg: Nature 314:472-474, 1985.) The present paper describes in detail the behaviour of profilactin and profilin in the presence of different types of phospholipids and neutral lipids under different conditions. PtdIns(4,5)P2 is the only phospholipid found so far which in the presence of 80 mM KCl and at Ca2+ concentrations below 10(-5) M effectively dissociates profilactin with the resulting polymerization of the actin. Phosphatidylinositol 4-monophosphate exhibits some activity but phosphatidylinositol is inactive. Both calf spleen profilin and profilin from human platelets form stable complexes with PtdIns(4,5)P2 micelles. PtdIns(4,5)P2 is active also when incorporated together with other phospholipids in mixed vesicles.     

Mango profilin: cloning,expression and cross-reactivity with birch pollen profilin Bet v 2

Mango can cause severe anaphylactic reactions. Profilin has been assumed partly responsible for the cross-reactivity between mango fruit and other allergens but has not been finally clarified. In this study, two isoforms of mango fruits profilin were amplified by RT-PCR and 3'RACE from total RNA. Each mango profilin cDNA includes an open reading frame coding for 131 amino acids. The deduced amino acid sequence of the corresponding protein show high identity with other allergenic profilins. Expression of the recombinant mango profilin was carried out in Escherichia coli BL21(DE3) using vector PET28a and the purification of the recombinant protein was performed via affinity chromatography with Ni+ coupled to sepharose. IgE reactivity of recombinant mango profilin was investigated by immunoblot and 8 of 18 mango-allergic patients tested presented specific IgE-antibodies to recombinant mango profilin. IgE-inhibition and ELISA inhibition experiments were performed to analyze mango profilin cross-reactivity with profilins from birch pollen and high cross-reactivities have been found.     

X-ray structure determination of human profilin II: A comparative structural analysis of human profilins

Human profilins are multifunctional, single-domain proteins which directly link the actin microfilament system to a variety of signalling pathways via two spatially distinct binding sites. Profilin binds to monomeric actin in a 1:1 complex, catalyzes the exchange of the actin-bound nucleotide and regulates actin filament barbed end assembly. Like SH3 domains, profilin has a surface-exposed aromatic patch which binds to proline-rich peptides. Various multidomain proteins including members of the Ena/VASP and formin families localize profilin:actin complexes through profilin:poly-L-proline interactions to particular cytoskeletal locations (e.g. focal adhesions, cleavage furrows). Humans express a basic (I) and an acidic (II) isoform of profilin which exhibit different affinities for peptides and proteins rich in proline residues. Here, we report the crystallization and X-ray structure determination of human profilin II to 2.2 A. This structure reveals an aromatic extension of the previously defined poly-L-proline binding site for profilin I. In contrast to serine 29 of profilin I, tyrosine 29 in profilin II is capable of forming an additional stacking interaction and a hydrogen bond with poly-L-proline which may account for the increased affinity of the second isoform for proline-rich peptides. Differential isoform specificity for proline-rich proteins may be attributed to the differences in charged and hydrophobic residues in and proximal to the poly-L-proline binding site. The actin-binding face remains nearly identical with the exception of five amino acid differences. These observations are important for the understanding of the functional and structural differences between these two classes of profilin isoforms.     

The proline-rich protein palladin is a binding partner for profilin

Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), alpha-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.     

Human profilin. Molecular cloning, sequence comparison, and chromosomal analysis

Profilin is an ubiquitous 12-15-kDa actin monomer-binding protein, the amino acid sequence of which was previously reported for the cow and Acanthamoeba. In the latter species, two isoforms of profilin have been identified. We have isolated full-length profilin cDNA clones from a human HepG2 library. All clones have the same nucleotide sequence, and Northern blot and RNase protection analyses of human tissues indicate that all tissues have the same approximately 850 base message, and provide no evidence of alternative message splicing. This result strongly implies a single profilin isoform in human cells, although differential post-translational modifications have not been excluded. Northern blot analysis extends the tissue distribution of profilin to include epithelial, muscle, and renal tissues. Comparison of the predicted human profilin amino acid sequence with that of published bovine profilin indicates 90% identity with a single 3-residue deletion in the human sequence. Southern blot analysis of somatic cell hybrid DNA indicates at least four dispersed genetic loci in the human genome hybridize with the profilin cDNA as well as untranslated region fragments, suggesting several of these loci represent pseudogenes of recent evolutionary origin. In addition, 5' and 3' untranslated regions are conserved between humans and rodents, implying a functional role for these regions of the profilin gene.     

Acanthamoeba profilin affects the mechanical properties of nonfilamentous actin

We investigated the mechanical properties of two abundant, cytoplasmic proteins from Acanthamoeba, profilin and actin, and found that while both profilin and nonfilamentous actin alone behaved as solids, mixtures of the two proteins were viscoelastic liquids. When allowed to equilibrate, profilin formed a viscoelastic solid with mechanical properties similar to filamentous and nonfilamentous actin. Consequently, profilin itself may contribute significantly to the elasticity and viscosity of cytoplasm. The addition of profilin to nonfilamentous actin caused a phase transition from gel (viscoelastic solid) to sol (viscoelastic liquid) when the concentration of free actin became too low to form a gel. In contrast, profilin had little effect on the rigidity and viscosity of actin filaments. We speculate that nonfilamentous actin and profilin, both of which form shear-sensitive structures, can be modeled as flocculant materials. We conclude that profilin may regulate the rigidity (elasticity) of the cytoplasm not only by inhibiting polymerization of actin, but also by modulating the mechanical properties of nonfilamentous actin.     

Primary structure of profilins from two species of Echinoidea and Physarum polycephalum

Profilin is a small G-actin-binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast. Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined. Two echinoid profilins were composed of 139 amino acid residues, N-termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE [Mabuchi, I. & Hosoya, H. (1982) Biomed. Res. 3, 465-476]. On the other hand, Physarum profilin was composed of 124 amino acid residues, the N-terminus was acylated, and the calculated molecular mass was 13132 Da. The sequences of C. japonicus and A. crassispina profilins were homologous (84% identical). However, the similarity of these profilins with those form other organisms was low. The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins. The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N-terminal region, which was suggested to be a common actin-binding region. The C-terminal region was also conserved, although to a lesser extent than the N-terminal region.     

Maize profilin isoforms are functionally distinct

Profilin is an actin monomer binding protein that, depending on the conditions, causes either polymerization or depolymerization of actin filaments. In plants, profilins are encoded by multigene families. In this study, an analysis of native and recombinant proteins from maize demonstrates the existence of two classes of functionally distinct profilin isoforms. Class II profilins, including native endosperm profilin and a new recombinant protein, ZmPRO5, have biochemical properties that differ from those of class I profilins. Class II profilins had higher affinity for poly-l-proline and sequestered more monomeric actin than did class I profilins. Conversely, a class I profilin inhibited hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate by phospholipase C more strongly than did a class II profilin. These biochemical properties correlated with the ability of class II profilins to disrupt actin cytoplasmic architecture in live cells more rapidly than did class I profilins. The actin-sequestering activity of both maize profilin classes was found to be dependent on the concentration of free calcium. We propose a model in which profilin alters cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration. These results provide strong evidence that the maize profilin gene family consists of at least two classes, with distinct biochemical and live-cell properties, implying that the maize profilin isoforms perform distinct functions in the plant.     

Distribution of profilin in fibroblasts correlates with the presence of highly dynamic actin filaments.

We have used polyclonal and monoclonal antibodies raised against calf thymus profilin to localize the corresponding protein in translocating, spreading, and stationary rat fibroblasts. Immunofluorescence of whole cells and immunogold labeling on ventral membranes of lysis-squirted cells showed that profilin was markedly enriched in the highly dynamic lamellipodia or pseudopodial lobes. Within these regions, a significant fraction was colocalized with dynamic actin filaments organized in actin ribs, cortical filaments, or stress fiber-like bundles, and little profilin was found in membrane areas appearing free of actin. In contrast, stress fibers of stationary cells as well as actin arcs and ring-like bundles of spreading and migrating cells showed very little label. These results are discussed in context with the proposed role of profilin in regional membrane dynamics typical for fibroblasts and are compared to previous data (Hartwig et al.: J. Cell Biol. 109:1571-1579, 1989) on profilin distribution in platelets and granulocytes.     

Purification and characterization of two isoforms of Acanthamoeba profilin

Acanthamoeba profilin purified according to E. Reichstein and E.D. Korn (1979, J. Biol. Chem. 254:6174-6179) consists of two isoforms (profilin- I and-II) with approximately the same molecular weight and reactivity to a monoclonal antibody but different isoelectric points and different mobilities on carboxymethyl-agarose chromatography and reversed-phase high-performance liquid chromatography. The isoelectric points of profilin-I is approximately 5.5 and that of profilin-II is greater than or equal to 9.0. Tryptic peptides from the two proteins are substantially different, which suggests that there are major differences in their sequences. At similar concentrations, both profilins prolong the lag phase at the outset of spontaneous polymerization and inhibit the extent of polymerization. Both forms also inhibit elongation weakly at the barbed end and strongly at the pointed end of actin filaments.