Profilin Isoforms Research Articles

Fine-tuning of neuronal architecture requires two profilin isoforms

Two profilin isoforms (PFN1 and PFN2a) are expressed in the mammalian brain. Although profilins are essential for regulating actin dynamics in general, the specific role of these isoforms in neurons has remained elusive. We show that knockdown of the neuron-specific PFN2a results in a significant reduction in dendrite complexity and spine numbers of hippocampal neurons. Overexpression of PFN1 in PFN2a-deficient neurons prevents the loss of spines but does not restore dendritic complexity. Furthermore, we show that profilins are involved in differentially regulating actin dynamics downstream of the pan-neurotrophin receptor (p75(NTR)), a receptor engaged in modulating neuronal morphology. Overexpression of PFN2a restores the morphological changes in dendrites caused by p75(NTR) overexpression, whereas PFN1 restores the normal spine density. Our data assign specific functions to the two PFN isoforms, possibly attributable to different affinities for potent effectors also involved in actin dynamics, and suggest that they are important for the signal-dependent fine-tuning of neuronal architecture.

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.

Testis-expressed profilins 3 and 4 show distinct functional characteristics and localize in the acroplaxome-manchette complex in spermatids

BackgroundMultiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin-1 (PFN1) and profilin-2 (PFN2).ResultsLigand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls. β-actin, phosphoinositides, poly-L-proline and mDia3, but not VASP, were confirmed as in vitro interaction partners of PFN3. In parallel experiments, PFN4 bound to selected phosphoinositides but not to poly-L-proline, proline-rich proteins, or actin. Immunofluorescence microscopy of PFN3 and PFN4 revealed distinct subcellular locations in differentiating spermatids. Both were associated first with the acroplaxome and later with the transient manchette. Predicted 3D structures indicated that PFN3 has the actin-binding site conserved, but retains only approximately half of the common poly-L-proline binding site. PFN4, in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data.ConclusionThe testis-specific isoform PFN3 showed major hallmarks of the well characterized "somatic" profilin isoforms, albeit with distinct binding affinities. PFN4, on the other hand, did not interact with actin or polyproline in vitro. Rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation.

In birds, profilin-2a is ubiquitously expressed and contributes to actin-based motility

Profilins are small actin-binding proteins expressed in all eukaryotes. They are involved in the regulation of actin filament dynamics and various signalling pathways. The identification of a variety of profilin isoforms led to the assumption that there may be isoform-specific functions. In mammals, profilin-1 (PFN1) is ubiquitously expressed and engaged in the regulation of various motility processes in all cell types. By contrast, profilin-2a (PFN2a) is mainly restricted to neuronal cells and there is evidence that it is involved in neuronal plasticity and membrane trafficking. However, the PFN2a sequence is much better conserved than PFN1 throughout different phyla, indicating that its restricted expression and specialized function in mammals might be unique. Using isoform-specific antibodies, we show that the situation is different in birds. PFN2a is ubiquitously expressed in embryonic and adult chicken tissues at equal and frequently higher amounts than in mammals. Together with PFN1, it is present in cultivated chicken fibroblasts, but differentially localized. Knockdown experiments with miRNA reveal that PFN2a is involved in cell adhesion, spreading and locomotion, and silencing this isoform has pronounced consequences on these processes. Our results indicate profilin isoform expression is differentially regulated among vertebrates.

Arabidopsis Profilin Isoforms, PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profilin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive localizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.

Formin Differentially Utilizes Profilin Isoforms to Rapidly Assemble Actin Filaments

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.

Molecular and immunological characterization of novel weed pollen pan‐allergens

Pan-allergens like profilins, calcium-binding proteins (CBPs), and nonspecific lipid transfer proteins have been suggested as possible specific markers for multiple pollen sensitizations, and could be used to predict cross-sensitization/poly-sensitization to several pollen allergens. Therefore, the purification and characterization of cross-reacting allergens in pollen is an extremely important task towards correct allergy diagnosis. New pan-allergens were identified by screening a ragweed pollen cDNA library with sera of patients allergic to mugwort pollen. Resulting proteins were cloned, expressed, purified and characterized. We report complete cDNA sequences of two profilin isoforms (Amb a 8.01 and Amb a 8.02), two isoforms of a 2EF-hand CBP (Amb a 9.01 and Amb a 9.02), a new 3EF-hand CBP (Amb a 10) from ragweed pollen and a 2EF-hand CBP from mugwort (Art v 5). All these proteins were expressed in Escherichia coli, purified to homogeneity and characterized by biochemical and immunological means. The identified proteins are novel pan-allergens and can be used as diagnostic markers for polysensitization and used in component-resolved diagnosis.

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.

Cloning and characterization of profilin (Pru du 4), a cross-reactive almond ( Prunus dulcis) allergen

The identity of allergenic almond proteins is incomplete. Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

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.

Caenorhabditis elegans expresses three functional profilins in a tissue‐specific manner

Profilins are actin binding proteins, which also interact with polyphosphoinositides and proline-rich ligands. On the basis of the genome sequence, three diverse profilin homologues (PFN) are predicted to exist in Caenorhabditis elegans. We show that all three isoforms PFN-1, PFN-2, and PFN-3 are expressed in vivo and biochemical studies indicate they bind actin and influence actin dynamics in a similar manner. In addition, they bind poly(L-proline) and phosphatidylinositol 4,5-bisphosphate micelles. PFN-1 is essential whereas PFN-2 and PFN-3 are nonessential. Immunostainings revealed different expression patterns for the profilin isoforms. In embryos, PFN-1 localizes in the cytoplasm and to the cell-cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in the neurons, the vulva, and the somatic gonad, PFN-2 in the intestinal wall, the spermatheca, and the pharynx, and PFN-3 localizes in a striking dot-like fashion in body wall muscle. Thus the model organism Caenorhabditis elegans expresses three profilin isoforms and is the first invertebrate animal with tissue-specific profilin expression.

Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin‐binding proteins and cause defects in cell expansion upon aberrant expression

The closely related proteins AtFH4 and AtFH8 represent the group Ie clade of Arabidopsis formin homologues. The subcellular localization of these proteins and their ability to affect the actin cytoskeleton were examined. AtFH4 protein activity was identified using fluorimetric techniques. Interactions between Arabidopsis profilin isoforms and AtFH4 were assayed in vitro and in vivo using pull-down assays and yeast-2-hybrid. The subcellular localization of group Ie formins was observed with indirect immunofluorescence (AtFH4) and an ethanol-inducible green fluorescent protein (GFP) fusion construct (AtFH8). AtFH4 protein affected actin dynamics in vitro, and yeast-2-hybrid assays suggested isoform-specific interactions with the actin-binding protein profilin in vivo. Indirect immunofluorescence showed that AtFH4 localized specifically to the cell membrane at borders between adjoining cells. Expression of an AtFH8 fusion protein resulted in GFP localization to cell membrane zones, similar to AtFH4. Furthermore, aberrant expression of AtFH8 resulted in the inhibition of root hair elongation. Taken together, these data suggest that the group Ie formins act with profilin to regulate actin polymerization at specific sites associated with the cell membrane.

Molecular cloning and characterization of an allergen from Persian melon

Rationale Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. Most of the melon-allergic patients (more than 70 percent) presented specific IgEantibodies to 14.4 Kda protein in melon extract that could be attributed to profilin. Methods To characterize allergenic component of the melon at the molecular level, this study was aimed at the cloning of melon major allergen cDNA. The IgE-binding component was isolated from melon by cation exchange FPLC and a purification step on poly-L-proline 4BSepharose. Cloning of melon profilin was performed by reverse transcription-PCR using degenerated primers. 5' and 3' RACE methods were carried out for the identification of the unknown 5'-end and 3'-end of the cDNA. Results Four isoforms of profilin identified that significant differences were noted in the non-coding regions, whereas coding sequence in one of them only showed a variation in position 54. Coding sequences of all isoforms (AY292385, AY292386, AY292385) had a length of 394 bp corresponding to 131 amino acid residues and a predicted isoelectric point of 4.46. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71-84%) recently described as allergens. Conclusions Profilin is an important allergenic compound in melon and shows high homology to pollens profilin. Pollenosis and associated allergies to melon can therefore partly be explained by the cross-reactivity between homologous profilins.

Molecular cloning and characterization of Persian melon major allergen (cuc m2)

Rationale Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. Most of the melon-allergic patients (more than 70 percent) presented specific IgEantibodies to 14.4 Kda protein in melon extract that could be attributed to profilin. Methods To characterize allergenic component of the melon at the molecular level, this study was aimed at the cloning of melon major allergen cDNA. The IgE-binding component was isolated from melon by cation exchange FPLC and a purification step on poly-L-proline 4BSepharose. Cloning of melon profilin was performed by reverse transcription-PCR using degenerated primers. 5' and 3' RACE methods were carried out for the identification of the unknown 5'-end and 3'-end of the cDNA. Results Four isoforms of profilin identified that significant differences were noted in the non-coding regions, whereas coding sequence in one of them only showed a variation in position 54. Coding sequences of all isoforms (AY292385, AY292386, AY292385) had a length of 394 bp corresponding to 131 amino acid residues and a predicted isoelectric point of 4.46. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71–84%) recently described as allergens. Conclusion Profilin is an important allergenic compound in melon and shows high homology to pollens profilin. Pollenosis and associated allergies to melon can therefore partly be explained by the cross-reactivity between homologous profilins.

Assessment of profilin as an allergen for latex-sensitized patients.

The presence of the actin-binding protein, profilin, has been demonstrated in natural latex extracts; but the clinical significance of this molecule as an allergen for latex-allergic patients is not clear. We studied the allergenic relevance of isolated latex natural and recombinant profilin, by in vivo and in vitro techniques, in two populations of spina bifida children (SB) and adults allergic to latex (AL). Profilin is present in small amounts in latex extracts obtained from low ammoniated (LA) natural latex. Its purification by affinity chromatography resulted difficult due to Heb v 1 unspecific binding. Therefore a method was developed to obtain natural profilin from natural latex, combining affinity chromatography (PLP, poly-L-proline Sepharose column) and previous ammonium sulfate fractionation. Alternatively, latex c-serum containing a low amount of Hev b 1 and a relatively higher profilin content could be used. Recombinant latex profilin isoform (rHev b 8) was cloned by PCR amplification. The entire coding region of Hev b 8 was subcloned into the expression vector pKN172 and a non-fusion form of Hev b 8 was expressed in Escherichia coli BL21 (DE3). Purified recombinant protein was obtained after a single passage through PLP-Sepharose column. Natural and recombinant purified Hev b 8 were tested cutaneously by intradermoreaction (ID) in 17 SB and 14 AL patients. They were positive in 15 SB and 14 AL patients. No wheals were produced when tested in nonatopic control patients. Only 42% of sera from latex-allergic patients revealed specific IgE titers of class 1 or higher by enzyme immunoassay and only 39% of them exhibited IgE binding by SDS-PAGE immunoblotting with any natural or recombinant Hev b 8 forms. It seems that profilin is a relevant allergen for both groups of patients from a frequency point of view, but with scarce presence in natural latex extracts and raw sources, with a subsequent low IgE induction capacity.

Molecular and immunological characterization of profilin from mugwort pollen.

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirty-six percent of the mugwort-allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgE-binding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollen-allergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgE-mediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE cross-reactivity of recombinant mugwort profilin and profilin from various pollen and food extracts.

Chlamydomonas reinhardtii produces a profilin with unusual biochemical properties.

We report the characterization of a profilin orthologue from Chlamydomonas reinhardtii. CrPRF, probably the only profilin isoform, is present in both the cell body and flagella. Examination of vegetative and gametic cells by immunofluorescence microscopy using multiple fixation procedures also revealed enrichment of CrPRF at the anterior of the cell near the base of flagella and near the base of the fertilization tubule in mating type plus gametes. Purified, recombinant CrPRF binds to actin with a Kd value approximately 10(-7) and displaces nuclei in a live cell 'nuclear displacement' assay, consistent with profilin's ability to bind G-actin in vivo. However, when compared with other profilin isoforms, CrPRF has a relatively low affinity for poly-L-proline and for phosphatidylinositol (4,5) bisphosphate micelles. Furthermore, and surprisingly, CrPRF inhibits exchange of adenine nucleotide on G-actin in a manner similar to human ADF or DNase I. Thus, we postulate that a primary role for CrPRF is to sequester actin in Chlamydomonas. The unusual biochemical properties of CrPRF offer a new opportunity to distinguish specific functions for profilin isoforms.

Profilin II is alternatively spliced, resulting in profilin isoforms that are differentially expressed and have distinct biochemical properties.

We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.

Alternative splicing of the mouse profilin II gene generates functionally different profilin isoforms.

Profilins are a conserved family of proteins participating in actin dynamics and cell motility. In the mouse, two profilin genes are known. Profilin I is expressed universally at high levels, while profilin II is expressed mainly in the brain. Here we describe the occurrence of two mouse profilin II isoforms, A and B, which are derived by alternative splicing. They are identical through residue 107 of the protein, but then have distinct C-terminal sequences. Profilin IIA binds to poly-L-proline and actin with high affinity similar to profilin I. Profilin IIB on the other hand does not bind to actin and the affinity for poly-L-proline is greatly diminished. However, tubulin was found to bind to GST-profilin IIB, and in vivo GFP-profilin IIB was recruited to spindles and asters during mitosis in HeLa cells. Our results indicate unexpected diversity in the functions of the profilin family of proteins, and suggest that in mouse profilin IIA is intimately involved in actin dynamics, while profilin IIB associates with other cytoskeletal components.

Maize Profilin Isoforms Are Functionally Distinct

Maize Profilin Isoforms Are Functionally Distinct