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 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.     

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.     

Profilin isoforms in Dictyostelium discoideum

Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.     

Characterization of renatured profilin purified by urea elution from poly-L-proline agarose columns

We present evidence that native profilin can be purified from cellular extracts of Acanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly-L-proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6-8 M. Acanthamoeba profilin-I and profilin-II can subsequently be separated by cation exchange chromatography. The yield of Acanthamoeba profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: 1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly-L-proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; 2) dialyzed profilins form 3-D crystals under the same conditions as native profilins; 3) dialyzed Acanthamoeba profilin-I has an NMR spectrum identical with that of native profilin-I; and 4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyostelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly-L-proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin-II or alpha-actinin in crude cell extracts can be bound selectively to the poly-L-proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin-II from both Acanthamoeba and Dictyostelium cell extracts and alpha-actinin from Acanthamoeba cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly-L-proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.     

Mouse profilin 2 regulates endocytosis and competes with SH3 ligand binding to dynamin 1

Mammalian profilins are abundantly expressed actin monomer-binding proteins, highly conserved with respect to their affinities for G-actin, poly-L-proline, and phosphoinositides. Profilins associate with a large number of proline-rich proteins; the physiological significance and regulation of which is poorly understood. Here we show that profilin 2 associates with dynamin 1 via the C-terminal proline-rich domain of dynamin and thereby competes with the binding of SH3 ligands such as endophilin, amphiphysin, and Grb2, thus interfering with the assembly of the endocytic machinery. We also present a novel role for the brain-specific mouse profilin 2 as a regulator of membrane trafficking. Overexpression of profilin 2 inhibits endocytosis, whereas lack of profilin 2 in neurons results in an increase in endocytosis and membrane recycling. Phosphatidylinositol 4,5-bisphosphate releases profilin 2 from the profilin 2-dynamin 1 complex as well as from the profilin 2-actin complex, suggesting that profilin 2 is diverging the phosphoinositide signaling pathway to actin polymerization as well as endocytosis.     

Oligomerization of profilins from birch, man and yeast. Profilin, a ligand for itself?

 Profilins are structurally well conserved low molecular weight (12–15 kDa) eukaryotic proteins which interact with a variety of physiological ligands: (1) cytoskeletal components, e.g., actin; (2) polyphosphoinositides, e.g., phosphatidylinositol-4,5-bisphosphate; (3) proline-rich proteins, e.g., formin homology proteins and vasodilatator-stimulated phosphoprotein. Profilins may thus link the microfilament system with signal transduction pathways. Plant profilins have recently been shown to be highly crossreactive allergens which bind to IgE antibodies of allergic patients and thus cause symptoms of type I allergy. We expressed and purified from Escherichia coli profilins from birch pollen (Betula verrucosa), humans (Homo sapiens) and yeast (Schizosaccharomyces pombe) and demonstrated that each of these profilins is able to form stable homo- and heteropolymers via disulphide bonds in vitro. Circular dichroism analysis of oxidized (polymeric) and reduced (monomeric) birch pollen profilin indicates that the two states have similar secondary structures. Using ¹²⁵I-labeled birch pollen, yeast and human profilin in overlay experiments, we showed that disulphide bond formation between profilins can be disrupted under reducing conditions, while reduced as well as oxidized profilin states bind to actin and profilin-specific antibodies. Exposure of profilin to oxidizing conditions, such as when pollen profilins are liberated on the surface of the mucosa of atopic patients, may lead to profilin polymerization and thus contribute to the sensitization capacity of profilin as an allergen. Received: 25 February 1998 / Revision accepted: 12 May 1998     

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.     

Profilin tyrosine phosphorylation in poly-L-proline-binding regions inhibits binding to phosphoinositide 3-kinase in Phaseolus vulgaris

The profilin family consists of a group of ubiquitous highly conserved 12-15 kDa eukaryotic proteins that bind actin, phosphoinositides, poly-l-proline (PLP) and proteins with proline-rich motifs. Some proteins with proline-rich motifs form complexes that have been implicated in the dynamics of the actin cytoskeleton and processes such as vesicular trafficking. A major unanswered question in the field is how profilin achieves the required specificity to bind such an array of proteins. It is now becoming clear that profilin isoforms are subject to differential regulation and that they may play distinct roles within the cell. Considerable evidence suggests that these isoforms have different functional roles in the sorting of diverse proteins with proline-rich motifs. All profilins contain highly conserved aromatic residues involved in PLP binding which are presumably implicated in the interaction with proline-rich motif proteins. We have previously shown that profilin is phosphorylated on tyrosine residues. Here, we show that profilin can bind directly to Phaseolus vulgaris phosphoinositide 3-kinase (PI3K) type III. We demonstrate that a new region around Y72 of profilin, as well as the N- and C-terminal PLP-binding domain, recognizes and binds PLP and PI3K. In vitro binding assays indicate that PI3K type III forms a complex with profilin in a manner that depends on the tyrosine phosphorylation status within the proline-rich-binding domain in profilin. Profilin-PI3K type III interaction suggests that profilin may be involved in membrane trafficking and in linking the endocytic pathway with actin reorganization dynamics.     

Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP.

The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks.     

Pollen profilin function depends on interaction with proline-rich motifs.

The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells. Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms. Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L-proline and monomeric actin from maize pollen. In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen. The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen. In contrast, the affinity of ZmPRO4 for poly-L-proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms. When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1. A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L-proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4. In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L-proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells.     

Functional characterization of green fluorescent protein-profilin fusion proteins.

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.     

Characterization of Ricinus communis phloem profilin,RcPRO1

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.     

Motility determinants in WASP family proteins

In response to upstream signals, proteins in the Wiskott-Aldrich Syndrome protein (WASP) family regulate actin nucleation via the Arp2/3 complex. Despite intensive study of the function of WASP family proteins in nucleation, it is not yet understood how their distinct structural organization contributes to actin-based motility. Herein, we analyzed the activities of WASP and Scar1 truncation derivatives by using a bead-based motility assay. The minimal region of WASP sufficient to direct movement was the C-terminal WCA fragment, whereas the corresponding region of Scar1 was insufficient. In addition, the proline-rich regions of WASP and Scar1 and the Ena/VASP homology 1 (EVH1) domain of WASP independently enhanced motility rates. The contributions of these regions to motility could not be accounted for by their direct effects on actin nucleation with the Arp2/3 complex, suggesting that they stimulate motility by recruiting additional factors. We have identified profilin as one such factor. WASP- and Scar1-coated bead motility rates were significantly reduced by depletion of profilin and VASP and could be more efficiently rescued by a combination of VASP and wild-type profilin than by VASP and a mutant profilin that cannot bind proline-rich sequences. Moreover, motility of WASP WCA beads was not affected by the depletion or addback of VASP and profilin. Our results suggest that recruitment of factors, including profilin, by the proline-rich regions of WASP and Scar1 and the EVH1 domain of WASP stimulates cellular actin-based motility.     

Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathways

Transformation progression of epithelial cells involves alterations in their morphology, polarity, and adhesive characteristics, all of which are associated with the loss and/or reorganization of actin structures. To identify the underlying mechanism of formation of the adhesion-dependent, circumferential actin network, the expression and localization of the actin binding and regulating proteins (ABPs), vinculin, VASP, and profilin were evaluated. Experimental depolarization of epithelial cells results in the loss of normal F-actin structures and the transient upregulation of vinculin, VASP, and profilin. This response is due to the loss of cell-cell, and not cell-substrate interactions, since cells that no longer express focal adhesions or stress fibers are still sensitive to changes in adhesion and manifest this in the altered profile of expression of these ABPs. Transient upregulation is dependent upon de novo protein synthesis, and protein kinase-, but not phosphatase-sensitive signal transduction pathway(s). Inhibition of the synthesis of these proteins is accompanied by dephosphorylation of the ribosomal S6 protein, but does not involve inhibition of the PI3-kinase-Akt-mTOR pathway. Constitutive expression of VASP results in altered cell morphology and adhesion and F-actin and vinculin structures. V12rac1 expressing epithelial cells are constitutively nonadhesive, malignantly transformed, and constitutively express high levels of these ABPs, with altered subcellular localizations. Transformation suppression is accompanied by the restoration of normal levels of the three ABPs, actin structures, adhesion, and epithelial morphology. Thus, vinculin, VASP, and profilin are coordinately regulated by signal transduction pathways that effect a translational response. Additionally, their expression profile maybe indicative of the adhesion and transformation status of epithelial cells.     

Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-L-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-L-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-L-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.     

Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin

A key event in signal transduction in many eukaryotes is activation of polyphosphoinositide-specific phospholipase C (PIC). This enzyme hydrolyses the plasma membrane-associated lipid, phosphatidylinositol(4,5)bisphosphate (Ptdlns(4,5)P₂) which leads to the production of the two second messenger molecules: inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃) and 1,2-diacylglycerol (DG). In plants, an enzyme which functionally resembles mammalian PIC is known to exist in the plasma membrane, but little is understood about how its activity is regulated. The recent discovery of several plant proteins with 30–40% homology to the mammalian actin- and phosphoinositide-binding protein, profilin, has prompted an investigation as to whether these proteins (plant profilins) are able to interact with polyphosphoinositides and, if so, whether such interactions have physiological relevance for signal transduction via the plant phosphoinositide system.
In this study it is demonstrated that a direct and highly specific interaction does exist between plant profilin and polyphosphoinositides and that these interactions dramatically affect the ability of plant plasma membrane phosphoinositide phospholipase C to utilize phosphoinositides for second messenger production. These data are the first to demonstrate a functional role of plant profilin in controlling polyphosphoinositide turnover and also provide the first evidence for a direct effect of an actin-binding protein on a membrane-associated signalling enzyme. These findings indicate a novel mechanism for control of plant phosphoinositide turnover, and suggest a possible link between plant cell activation, second messenger production and modulation of cytoskeletal dynamics.     

The profilin:actin complex localizes to sites of dynamic actin polymerization at the leading edge of migrating cells and pathogen-induced actin tails

A unique set of affinity-purified anti-profilin and anti-actin antibodies generated against a covalently coupled version of the profilin:actin complex was used to assess the distribution of profilin and non-filamentous actin in mouse melanoma cells. In agreement with the profilin:actin complex being the principal source of actin for filament formation, we observed extensive co-distribution of both antibody preparations with vasodilator-stimulated phosphoprotein (VASP) and the p34 subunit of the Arp2/3 complex, both of which are components of actin polymer-forming protein complexes in the cell. This suggests that the localization of profilin and actin revealed with these antibodies in fact reflects the distribution of the profilin:actin complex rather than the two proteins separately. Significantly, protruding lamellipodia and filopodia showed intensive labeling. The two antibody preparations were also used to stain HeLa cells infected with Listeria monocytogenes or vaccinia virus. In both cases, the pattern of antibody staining of the pathogen-induced microfilament arrangement differed, suggesting a varying accessibility for the antibody-binding epitopes.     

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.     

Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.