Polyproline Binding Research Articles

Structural Characterization of a Thorarchaeota Profilin Indicates Eukaryotic-Like Features but with an Extended N-Terminus.

The emergence of the first eukaryotic cell is preceded by evolutionary events, which are still highly debatable. Clues of the exact sequence of events are beginning to emerge. Recent metagenomics analyses has uncovered the Asgard super-phylum as the closest yet known archaea host of eukaryotes. Some of these have been tested and confirmed experimentally. However, the bulk of eukaryotic signature proteins predicted to be encoded by the Asgard super-phylum have not been studied, and their true functions, at least in the context of a eukaryotic cell, are still elusive. For example, there are several different variants of the profilin within each Asgardian Achaea, and there are some conflicting results of their actual roles. Here, the 3D structure of profilin from Thorarchaeota is determined by nuclear magnetic resonance spectroscopy and shows that this profilin has a eukaryotic-like profilin with a rigid core and an extended N-terminus previously implicated in polyproline binding. In addition, it is also shown that Thorarchaeota Profilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirms the notion that Asgardian encoded proteins possess eukaryotic-like characteristics and strengthen the likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor.

Heimdallarchaea encodes profilin with eukaryotic-like actin regulation and polyproline binding

It is now widely accepted that the first eukaryotic cell emerged from a merger of an archaeal host cell and an alphaproteobacterium. However, the exact sequence of events and the nature of the cellular biology of both partner cells is still contentious. Recently the structures of profilins from some members of the newly discovered Asgard superphylum were determined. In addition, it was found that these profilins inhibit eukaryotic rabbit actin polymerization and that this reaction is regulated by phospholipids. However, the interaction with polyproline repeats which are known to be crucial for the regulation of profilin:actin polymerization was found to be absent for these profilins and was thus suggested to have evolved later in the eukaryotic lineage. Here, we show that Heimdallarchaeota LC3, a candidate phylum within the Asgard superphylum, encodes a putative profilin (heimProfilin) that interacts with PIP2 and its binding is regulated by polyproline motifs, suggesting an origin predating the rise of the eukaryotes. More precisely, we determined the 3D-structure of Heimdallarchaeota LC3 profilin and show that this profilin is able to: i) inhibit eukaryotic actin polymerization in vitro; ii) bind to phospholipids; iii) bind to polyproline repeats from enabled/vasodilator‐stimulated phosphoprotein; iv) inhibit actin from Heimdallarchaeota from polymerizing into filaments. Our results therefore provide hints of the existence of a complex cytoskeleton already in last eukaryotic common ancestor.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR–SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

A Fluorophore Fusion Construct of Human Profilin I with Non-Compromised Poly(L-Proline) Binding Capacity Suitable for Imaging

Profilin is vital for actin organisation in eukaryotic cells. It controls actin filament formation by binding monomeric actin and numerous proteins involved in polarised actin assembly. Important for the latter is the interaction surface formed by the N- and C-terminal helices, which pack close to each other on one side of the molecule at a distance from the actin site and mediate binding to poly-proline sequences present in many of the targeted proteins. Via these interactions, profilin contributes to the spatiotemporal control of actin filament growth. Studies of profilin dynamics in living cells by imaging techniques have been hampered by problems to generate fusion constructs with fluorophore proteins without negatively impacting on its poly-proline binding. With the object to circumvent this problem, we have generated an internal fusion of profilin with the green fluorescent variant citrine, here referred to as citrine–profilin. The characterisation of citrine–profilin (CIT-Pfn) demonstrates that it has full capacity to interact with poly-proline and also binds phosphatidylinositol lipids and actin, albeit with 10 times reduced affinity for the latter. Imaging of living cells expressing CIT-Pfn showed a distribution of the fusion protein similar to endogenous profilin. Furthermore, CIT-Pfn rescued the phenotypes observed after the Crispr/Cas9 knockout of the profilin 1 gene, including the lost migratory capacity characterising the knockout cells. Based on this, we conclude that the CIT-Pfn construct will be useful as a tool for displaying profilin localisation in living cells and obtaining information on its dynamic organisation under different conditions and activations of the actin microfilament and microtubule systems.

IQGAP1 regulates actin cytoskeleton organization in podocytes through interaction with nephrin

Increasing data has shown that the cytoskeletal reorganization of podocytes is involved in the onset of proteinuria and the progression of glomerular disease. Nephrin behaves as a signal sensor of the slit diaphragm to transmit cytoskeletal signals to maintain the unique structure of podocytes. However, the nephrin signaling cascade deserves further study. IQGAP1 is a scaffolding protein with the ability to regulate cytoskeletal organization. It is hypothesized that IQGAP1 contributes to actin reorganization in podocytes through interaction with nephrin. IQGAP1 expression and IQGAP1-nephrin colocalization in glomeruli were progressively decreased and then gradually recovered in line with the development of foot process fusion and proteinuria in puromycin aminonucleoside-injected rats. In cultured human podocytes, puromycin aminonucleoside-induced disruption of F-actin and disorders of migration and spreading were aggravated by IQGAP1 siRNA, and these effects were partially restored by a wild-type IQGAP1 plasmid. Furthermore, the cytoskeletal disorganization stimulated by cytochalasin D in COS7 cells was recovered by cotransfection with wild-type IQGAP1 and nephrin plasmids but was not recovered either by single transfection of the wild-type IQGAP1 plasmid or by cotransfection of mutant IQGAP1 [△1443(S→A)] and wild-type nephrin plasmids. Co-immunoprecipitation analysis using lysates of COS7 cells overexpressing nephrin and each derivative-domain molecule of IQGAP1 demonstrated that the poly-proline binding domain and RasGAP domain in the carboxyl terminus of IQGAP1 are the target modules that interact with nephrin. Collectively, these findings showed that activated IQGAP1, as an intracellular partner of nephrin, is involved in actin cytoskeleton organization and functional regulation of podocytes.

Integrin αIIbβ3-Mediated c-Src Activation: Differential Binding to Inactive and Active c-Src

Activation of the tyrosine kinase c-Src is a fundamental element of integrin αIIbβ3-mediated outside-in signaling in platelets. Presently, it is thought that c-Src is bound via its SH3 domain to the C-terminal Arg-Gly-Thr (RGT) sequence of the β3 cytoplasmic tail (CT). Although a recent NMR study supports this contention, it is likely that such binding would be precluded in the inactive conformation of c-Src because the linker connecting the SH2 and kinase domains of c-Src physically occludes the putative β3 CT binding site in the SH3 domain. Accordingly, we have re-examined the interaction between c-Src and β3 by immunoprecipitating β3 bound c-Src from resting and agonist-stimulated platelets and then applied the biophysical techniques of surface plasmon resonance (SPR) and NMR spectroscopy to directly characterize the interaction of the SH3 domain with the β3 CT. In unstimulated platelets, there was little or no association between c-Src and β3. However, following platelet stimulation with 1 unit/ml thrombin, c-Src binding to β3 is detectable within 10 sec of stimulation and binding persists for at least the next 90 sec. Phosphorylation of c-Src residue Tyr419 located in its activation loop is an indicator of c-Src activation. We detected the phosphorylation of β3-bound c-Src on Tyr419 at 20 sec following thrombin stimulation. Tyr419 phosphorylation then persisted for the next 40 sec after which it rapidly declined. Thus, platelet stimulation induces the rapid interaction of c-Src with the β3 subunit of αIIbβ3 and this interaction is accompanied by the induction of c-Src kinase activity. Since it has been reported that c-Src binds to β3 via its SH3 domain, we used SPR spectroscopy to measure the strength of this interaction. In initial experiments, we measured binding of the C-terminal β3 heptapeptide NITYRGT to the immobilized SH3 domain and detected NITYRGT binding with a dissociation constant (Kd) of approximately 700 mM. We then repeated the measurements by flowing the SH3 domain over the entire 43 residue β3 CT appended to the SPR chip. Under these conditions, we detected a Kd for SH3 domain binding to the β3 CT of 7.21 ± 2.42 µM. Previously, we reported similar behavior for talin-1 FERM domain binding to the β3 CT. Thus, these experiments suggest that like binding of the talin-1 FERM domain to the β3 CT, the interaction of c-Src with β3 is a two-dimensional ternary interaction in which the membrane surface makes an important contribution. Next, to identify the sites in the SH3 domain that interact with β3, we used NMR spectroscopy. In these experiments, we measured the interaction of the β3 peptide NITYRGT with the 15N-labeled SH3 domain. Because NITYRGT binding to the SH3 domain is weak, we used chemical shift perturbations (CSP) to identify the SH3 domain residues interacting with NITYRGT. We found that NITYRGT interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 CT (NITYEGK) induced only small CSP on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT-loop. Under these conditions, the β3 peptide induced CSP similar to the negative control. Thus, these studies indicate that the primary interaction of c-Src with the β3 CT occurs in its activated state at a site that overlaps with the polyproline binding site in its SH3 domain and suggest that protein-membrane interactions make an important contribution to the strength of binding. By contrast, interactions of inactive c-Src with β3 are weak and insensitive to β3 CT mutations. DisclosuresNo relevant conflicts of interest to declare.

The adapter protein CD2AP binds to p53 protein in the cytoplasm and can discriminate its polymorphic variants P72R.

Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour-suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R.

Analysis of β3 Binding to the c-Src SH3 Domain

AbstractAbstract 383An essential component of αIIbβ3-mediated outside-in signaling is activation of the tyrosine kinase c-Src, some of which is constitutively bound via its SH3 domain to the C-terminal Arg759-Gly760-Thr761 (RGT) sequence of the β3 cytoplasmic tail. RGT is quite different from the canonical polyproline sequence recognized by SH3 domains in which a polyproline helix packs against a shallow groove composed of aromatic residues (Tyr93, Tyr95, Tyr139 in c-Src). A specificity pocket located at the end of the groove and composed of residues from the n-Src- and RT-loops affects substrate specificity. Because of the obvious difference between RGT and polyproline sequences, we asked how RGT binds to the c-Src SH3 domain and what implications this has for c-Src regulation by αIIbβ3. Initially, we employed CD spectroscopy and tryptophan (Trp) fluorescence because these techniques are sensitive to changes in the local environment surrounding aromatic residues. However, there were no differences in the CD spectrum of the SH3 domain in absence or presence of the β3 peptide NITYRGT, whereas there was a clear shift in the presence of the core polyproline peptide RPLPPLP. Polyproline binding to Trp in the SH3 specificity pocket also results in a blue shift in Trp fluorescence from 355 nm to 347 nm; however, the fluorescence spectrum was essentially unchanged in the presence of NITYRGT. These experiments suggest that either the interaction of NITYRGT with SH3 is extremely weak and not observed at the concentrations used or occurs outside of the aromatic groove and the specificity pocket. Accordingly, we turned to NMR, a method able to detect weak protein-protein interactions. Two dimensional 1H-15N HSQC spectra of the SH3 domain in the presence of NITYRGT exhibited a number of changes in chemical shift compared to the spectrum in the absence of ligand. Sixteen residues located in the n-Src and RT-loops, grouped around the specificity pocket, had chemical shift changes > 0.05 ppm. The largest changes occurred in residues in or adjacent to the RT-loop, especially residues Arg98, Glu100, and Asp102. Of the resides forming the aromatic groove, only Tyr95 which is adjacent to the specificity pocket was perturbed by NITYRGT. Plots of the chemical shift changes for NH groups in SH3 vs. NITYRGT concentration were linear, indicating that the majority of SH3 domain was unbound. Further, a Kd for NITYRGT binding to SH3, estimated from these experiments, was between 175–350 mM. Next, we obtained HSQC spectra for SH3 in the presence of either RPLPPLP or a negative control peptide NITYEGK. Major perturbations due to RPLPPLP occurred in three regions: residues 98–103 (RT-loop), 116–122 (n-Src loop and specificity pocket), and residues 134–138; residues in the aromatic cluster were unaffected by the ligand. By contrast, only a handful of residues showed small perturbations in the presence of NITYEGK and there was no overlap between the affected residues and those affected by RPLPPLP. In conclusion, our results indicate that compared to polyproline sequences, the C-terminus of the β3 cytoplasmic tail binds to the c-Src SH3 domain in the region of the SH3 specificity pocket. Because chemical shifts for acidic residues located in the RT-loop were particularly sensitive to the presence of NITYRGT, it is likely that Arg759 in β3 makes an important contribution to the interaction. Moreover, we found that the interaction between NITYRGT and the c-Src SH3 domain is substantially weaker than was previously reported for the interaction of β3 with c-Src. This suggest the possibility that a third component is required for this interaction to occur under biological conditions. Recently we found that the β3 cytoplasmic tail in solution has weak affinity for the talin-1 FERM domain, but appending the tail to acidic phospholipids increased its affinity by three orders of magnitude. Since the c-Src SH3 domain contains a conserved patch of basic residues that are necessary for binding to acidic phospholipids, it is possible that the interaction of c-Src with β3 is also a ternary interaction in which protein-lipid interactions play an important role. Disclosures:No relevant conflicts of interest to declare.

Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: Intermolecular bivalent binding

The Grb2 adapter protein is involved in the activation of the Ras signaling pathway. It recruits the Sos protein by binding of its two SH3 domains to Sos polyproline sequences. We observed that the binding of Grb2 to a bivalent ligand, containing two Sos-derived polyproline-sequences immobilized on a SPR sensor, shows unusual kinetic behavior. SPR-kinetic analysis and supporting data from other techniques show major contributions of an intermolecular bivalent binding mode. Each of the two Grb2 SH3 domains binds to one polyproline-sequence of two different ligand molecules, facilitating binding of a second Grb2 molecule to the two remaining free polyproline binding sites. A molecular model based on the X-ray structure of the Grb2 dimer shows that Grb2 is flexible enough to allow this binding mode. The results fit with a role of Grb2 in protein aggregation, achieving specificity by multivalent interactions, despite the relatively low affinity of single SH3 interactions.

Expression analysis of Entamoeba invadens profilins in encystation and excystation

Cell motility by actin cytoskeleton is essential for differentiation processes of excystation and encystation of Entamoeba. We recently studied an actin depolymerizing factor (ADF)/cofilin (Cfl) of Entamoeba invadens (Ei), and demonstrated its contribution to the encystation and excystation of E. invadens through actin cytoskeletal reorganization. Profilin is also an actin-binding protein but its function is different from that of Cfl in actin assembly. This study investigated E. invadens profilins in relation to encystation and excystation which were induced in axenic culture systems. A homology search of the E. invadens genome database and molecular cloning identified four profilins of the parasite named EiPFN1, EiPFN2, EiPFN3, and EiPFN4. There were also multiple genes of profilin in Entamoeba histolytica (Eh) and Entamoeba dispar (Ed), each of which had three profilins. A search for conserved domains revealed that these profilins of Entamoeba had actin, phosphoinositide, and poly-proline binding sites. Phylogenetic analysis revealed that EiPFN3 and EiPFN4 formed the same clades including EhPFN3 and EdPFN3, and EhPFN2 and EdPFN2, respectively, while EiPFN1 and EiPFN2 were separated from EhPFN1 and EdPFN1. Rabbit anti-EiPFN1 serum reacted with recombinant EiPFN3 and EiPFN4 but not EiPFN2, and also reacted with EiPFN in lysates of cysts and trophozoites. Immunofluorescence staining with this antiserum showed co-localization of EiPFN with actin beneath the cell membrane through the life stages and also showed cytoplasmic localization. Both proteins proved to be rich in pseudopodia of trophozoites. Real-time RT-PCR showed that the mRNA level of EiPFN1 and EiPFN4 in trophozoites was comparable but that of EiPFN2 and EiPFN3 was very low. During encystation, the mRNA expression of EiPFN1 and EiPFN4 increased remarkably in the early phase much higher than that of EiPFN2 and EiPFN3. Then, the expression of all four PFNs sharply decreased in the later phase. This was in contrast to the sharp decrease in the mRNA level of EiCfl-2 during encystation in our previous study. In cysts, EiPFN1 was most abundantly expressed and EiPFN4 was at a lower level, while the expressions of EiPFN2 and EiPFN3 were virtually absent. Following the induction of excystation, mRNA levels of EiPFN1, EiPFN2, and EiPFN4 in cysts 5 h after induction were significantly higher than those in cysts before induction, while that of EiPFN3 was slightly higher than before induction. The mRNAs of EiPFN1 increased most extensively when the excystation was induced in the presence of cytochalasin D. Small interfering RNA (siRNA) to EiPFN1 inhibited both encystation and excystation but not growth. These findings demonstrate different expression of EiPFNs and the contribution of EiPFN to the encystation and excystation.

The solution structure of pGolemi, a high affinity Mena EVH1 binding miniature protein, suggests explanations for paralog-specific binding to Ena/VASP homology (EVH) 1 domains

Ena/VASP homology 1 (EVH1) domains are polyproline binding domains that are present in a wide range of adaptor proteins, among them Ena/VASP proteins involved in actin remodeling and axonal guidance. The interaction of ActA, a transmembrane protein from the food-borne pathogen Listeria monocytogenes, with EVH1 domains has been shown to be crucial for recruitment of the host's actin skeleton and, as a consequence, for the infectivity of this bacterium. We present the structure of a synthetic high-affinity Mena EVH1 ligand, pGolemi, capable of paralog-specific binding, solved by NMR spectroscopy. This peptide shares the common pancreatic peptide fold with its scaffold, avian pancreatic peptide, but shows pivotal differences in the amino-terminus. The interplay of spatial fixation and flexibility appears to be the reason for its high affinity towards Mena EVH1. Combined with earlier investigations, our structural data shed light on the specificity determinants of pGolemi and the importance of additional binding epitopes around the residues Thr74 and Phe32 on EVH1 domains regulating paralog specificity. Our results are expected to facilitate the design of other high-affinity, paralog-specific EVH1 domain ligands, and serve as a fundament for the investigation of the molecular mode of action of EVH1 domains.

Characterization of the Weak Interaction of C-Src with the β3 Cytoplasmic Tail

Integrins are ubiquitously expressed α/β heterodimers that mediate cell-cell and cellextracellular matrix interactions. Integrins reside on the plasma membrane in a highly regulated dynamic equilibrium between an inactive resting state and an active ligand binding conformation. An essential feature of this equilibrium is the association and dissociation of integrin transmembrane (TM) and cytoplasmic domains. Thus, when integrins are inactive, the TM and cytoplasmic domains of their α and β subunits are in proximity; the domains separate when integrins assume their active conformations. Subsequent to ligand binding, integrins cluster to generate “outside-in” signals, believed to be initiated by the activation of integrin-associated c-Src and related kinases. The prototypic example of regulated integrin activation and outside-in signaling is the major platelet integrin αIIbβ3. We have employed exogenous αIIb TM binding peptides to stabilize the separated conformation of αIIbβ3 and better understand the link between integrin conformation and downstream signal transduction. These peptides activate αIIbβ3 independent of “inside-out” signal transduction and induce αIIbβ3-mediated platelet aggregation despite the presence of the inhibitory prostaglandin PGE1 or the metabolic inhibitors 2-deoxyglucose and NaN3. When platelets are stimulated by natural agonists such as ADP or thrombin, fibrinogen binds to αIIbβ3 after which the c-Src associated with the β3 cytoplasmic tail is activated. This results in a characteristic phosphotyrosine signal that can be detected by immunoblotting platelet lysates using phosphotyrosine-specific antibodies and can be inhibited by the αIIbβ3 antagonists abciximab and RGDS that inhibit fibrinogen binding and clustering of αIIbβ3 in the membrane. By contrast, exogenous αIIb TM binding peptides activate phosphotyrosine signaling in platelets in the presence of abciximab and RGDS. This clustering-independent signal transduction is both integrin- and Src-dependent as it is absent in β3 knockout mice and can be blocked by the Src inhibitors PP2 and dasatinib. To better understand how β3-associated Src kinases are activated, we investigated the interaction of c-Src with αIIbβ3 by NMR spectroscopy. It has been reported that the SH3 domain of c-Src interacts with the C-terminal three amino acids of β3, Arg-Gly-Thr. We synthesized short fragments of the β3 cytoplasmic domain containing this sequence and observed perturbations in the 15N-1H HSQC NMR spectrum of the c-Src SH3 domain. There were significant shifts in the backbone amide spectra for residues Arg19, Asp23, Leu24, Glu21, Thr20, Tyr55, and Asn36, as well as shifts in the spectra for the side chains of Trp42, Trp43, Asn37, and Asn59. Titration experiments revealed a dissociation constant of approximately 4 mM for β3 peptide binding to the SH3 domain. It is noteworthy that the perturbed residues are localized to one region of the SH3 domain and are located outside of the canonical polyproline binding groove, implying a new mode of SH3 interaction. Moreover, the weak interaction of c-Src with the β3 cytoplasmic domain is consistent with the high local concentrations of β3 and Src kinases at the platelet membrane and the dynamic nature of integrin protein-protein interactions. These results suggest that the cytoplasmic domain separation that accompanies αIIbβ3 activation may be sufficient to activate the Src kinases weakly associated with β3 cytoplasmic domain. Subsequent αIIbβ3 clustering and/or c-Src dissociation may then amplify the tyrosine phosphorylation signal to complete outside-in signaling.

Profilin induces lamellipodia by growth factor independent mechanism

Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time- and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.

Vascular Hypertrophy and Hypertension Caused by Transgenic Overexpression of Profilin 1

We have overexpressed either the cDNA of human profilin 1 or expressed the mutant (88R/L) in the blood vessels of transgenic FVB/N mice. Reverse transcription-PCR indicated selective overexpression of profilin 1 and 88R/L in vascular smooth muscle cells. Polyproline binding showed increased profilin 1 and 88R/L proteins in transgenic mice compared with control (~30%, p < 0.05). Rhodamine-phalloidin staining revealed increase stress fiber formation in vascular smooth muscle cells of profilin 1 compared with 88R/L and control. Hematoxylin and eosin staining showed clear signs of vascular hypertrophy in the aorta of profilin 1 mice versus 88R/L and control. However, there were no differences between 88R/L and control mice. Western blotting confirmed the activation of the hypertrophic signaling cascades in aortas of profilin 1 mice. Phospho-ERK1/2 was significantly higher in profilin 1 than 88R/L and control (512.3 and 361.7%, respectively, p < 0.05). Profilin 1 mice had significant increases in phospho-JNK as compared with 88R/L and control (371.4 and 346%, respectively, p < 0.05). However, there were no differences between 88R/L and control mice in both kinases. There was a significant increase in ROCK II kinase in the aorta of profilin 1 mice compared with controls (>400%, p < 0.05). Tail cuff and circadian monitoring of blood pressure showed significant increases in systolic and mean arterial blood pressures of profilin 1 mice starting at age 6 months compared with controls (~25 mm Hg, p < 0.05). These results suggest that increased actin polymerization in blood vessels triggers activation of the hypertrophic signaling cascades and results in elevation of blood pressure at advanced age.

SH3-Hunter: discovery of SH3 domain interaction sites in proteins

SH3-Hunter (http://cbm.bio.uniroma2.it/SH3-Hunter/) is a web server for the recognition of putative SH3 domain interaction sites on protein sequences. Given an input query consisting of one or more protein sequences, the server identifies peptides containing poly-proline binding motifs and associates them to a list of SH3 domains, in order to compose peptide–domain pairs. The server can accept a list of peptides and allows users to upload an input file in a proper format. An accurate selection of SH3 domains is available and users can also submit their own SH3 domain sequence.SH3-Hunter evaluates which peptide–domain pair represents a possible interaction pair and produces as output a list of significant interaction sites for each query protein. Each proposed interaction site is associated to a propensity score and sensitivity and precision levels for the prediction. The server prediction capability is based on a neural network model integrating high-throughput pep-spot data with structural information extracted from known SH3-peptide complexes.

Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.

Structure de la sous-unité β du canal calcique : la place du β-interaction domain

Voltage-gated calcium channels are key players in a number of fundamental physiological functions including contraction, secretion, transmitter release or gene activation. They allow a flux of calcium into the cell that constitutes a switch-on signal for most of these functions. The structures responsible for the shaping of these fluxes by the membrane voltage belong to the channel itself, but a number of associated proteins are known to more precisely tune this calcium entry and adapt it to the cellular demand. The calcium channel regulatory beta subunit is undoubtedly the most important one, being influent on the expression, the kinetics, the voltage-dependence of channel opening and closing and on the pharmacology of the channel. Heterologous expression, combined to mutagenesis and electrophysiological and biochemical experiments have revealed the roles of short sequences of the beta subunit, including the BID (beta-interaction domain), in the physical and functional interactions with the channel pore. The resolved crystal structure of the beta subunit now sheds new light on these sequences and their interactions with the rest of the protein. The presence of a type 3 src-homology (SH3) domain and a guanylate kinase (GK) domain confirms that the subunit belongs to the MAGUK protein family. Consistently, the polyproline binding site and the kinase function of the SH3 and the GK domains, respectively, are non functional, and the BID appears to be buried in the structure, preserving the SH3-GK interaction but not directly available for interactions with the channel pore subunit. Anchoring of the beta subunit to the channel occurs via a hydrophobic grove in the GK domain, leaving a large surface of the subunit open to other protein-protein interactions. To what extent the intramolecular SH3-GK interaction is necessary for the stabilisation of this grove in a functional unit remains to be understood. The beta subunit may thus play a key role in scaffolding multiple proteins around the channel and organizing diverse calcium-dependent signalling pathways directly linked to voltage-gated calcium entry. These findings will undoubtedly vitalize the search for new beta-specific partners and functions.

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.

Polyproline binding is an essential function of human profilin in yeast.

Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP2) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1Delta. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP2. These data strongly suggest that polyproline binding is an essential function of profilin.

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.