Proline-rich Stretch Research Articles

A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition.

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

PAQR9 Modulates BAG6-mediated protein quality control of mislocalized membrane proteins.

Protein quality control is crucial for maintaining cellular homeostasis and its dysfunction is closely linked to human diseases. The post-translational protein quality control machinery mainly composed of BCL-2-associated athanogene 6 (BAG6) is responsible for triage of mislocalized membrane proteins (MLPs). However, it is unknown how the BAG6-mediated degradation of MLPs is regulated. We report here that PAQR9, a member of the Progesterone and AdipoQ receptor (PAQR) family, is able to modulate BAG6-mediated triage of MLPs. Analysis with mass spectrometry identified that BAG6 is one of the major proteins interacting with PAQR9 and such interaction is confirmed by co-immunoprecipitation and co-localization assays. The protein degradation rate of representative MLPs is accelerated by PAQR9 knockdown. Consistently, the polyubiquitination of MLPs is enhanced by PAQR9 knockdown. PAQR9 binds to the DUF3538 domain within the proline-rich stretch of BAG6. PAQR9 reduces the binding of MLPs to BAG6 in a DUF3538 domain-dependent manner. Taken together, our results indicate that PAQR9 plays a role in the regulation of protein quality control of MLPs via affecting the interaction of BAG6 with membrane proteins.

Differential effects of soluble and aggregating polyQ proteins on cytotoxicity and type-1 myosin-dependent endocytosis in yeast

Huntington’s disease develops when the polyglutamine (polyQ) repeat in the Huntingtin (Htt) protein is expanded to over 35 glutamines rendering it aggregation-prone. Here, using Htt exon-1 as a polyQ model protein in a genome-wide screen in yeast, we show that the normal and soluble Htt exon-1 is toxic in cells with defects in type-1 myosin-dependent endocytosis. The toxicity of Htt is linked to physical interactions with type-1 myosins, which occur via the Htt proline-rich region, leading to a reduction in actin patch polarization and clathrin-dependent endocytosis. An expansion of the polyQ stretch from 25 to 103 glutamines, which causes Htt aggregation, alleviated Htt toxicity in cells lacking Myo5 or other components involved in early endocytosis. The data suggest that the proline-rich stretch of Htt interacts with type-1 myosin/clathrin-dependent processes and demonstrate that a reduction in the activity of such processes may result in a positive selection for polyQ expansions.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

Profilin Binding Modulates the Aggregation and Phase Separation of Huntingtin N-Terminal Fragments via Polyphasic Linkage

Neurotoxic aggregates formed by N-terminal fragments of the mutant huntingtin protein (Htt-NTFs) are associated with the onset and progression of Huntington's disease. Htt-NTFs encompass expanded polyglutamine (polyQ) tracts that are flanked on the N-terminus by a 17-residue stretch and on the C-terminus by a 38-residue proline-rich stretch (C38). Htt-NTF aggregation and toxicity in cells is diminished by direct interactions with profilin, which is an abundant actin-binding protein that also binds polyproline ligands through a distinct binding site.Htt-NTF aggregation leads to the formation of soluble spherical aggregates (30-50 nm in diameter) and insoluble fibrillar deposits. These aggregates constitute distinct phases and are characterized by saturation concentrations, i.e., threshold concentrations that must be crossed in order for the corresponding phases to form. Using a combination of in vitro measurements we have demonstrated that profilin binding lowers the driving forces for Htt-NTF aggregation and phase separation via polyphasic linkage, a phenomenon whereby distinct saturation concentrations are shifted upwards by an order of magnitude. Accordingly, in the presence of profilin, a higher total concentration of Htt-NTFs is required to cross a given phase boundary. Our binding assays reveal that profilin binds preferentially to Htt-NTF monomers and small soluble oligomers. This involves specific interactions between profilin and the polyproline modules of C38 and possibly non-specific interactions between profilin and polyQ via an avidity effect.Our data provide a biophysical explanation for the observed cellular effects of profilin on Htt-NTF aggregation. Increases in saturation concentrations through polyphasic linkage might be a general mechanism for lowering the driving forces for aggregation and phase separation of aggregation-prone proteins, and may contribute to the protein homeostasis mechanisms used by cells to cope with protein aggregation stress.

Multiscale Simulations Provide Mechanistic Insights into the Effects of Sequence Contexts on Early-Stage Polyglutamine-Mediated Aggregation

Huntington's disease is associated with an expanded polyglutamine (polyQ) tract in huntingtin. The relevant aggregation prone toxic species encompasses an N-terminal 17-residue amphipathic stretch (N17), the polyQ tract, and a C-terminal 38-residue proline-rich stretch (C38). Experiments have shown that C38 decreases the overall driving force to form aggregates, whereas N17 decreases the overall solubility and accelerates fibril formation by destabilizing non-fibrillar species. We present results from multiscale simulations to explain how N17 and C38 accomplish their distinct modulatory behaviors. Our inverse Boltzmann procedure yields a systematic coarse-graining method whereby we bootstrap against atomistic simulations of monomers and reversible associations for pairs of molecules. We demonstrate that the species distributions and kinetics of forming early-stage aggregates are governed by the relative favorability of intermolecular interactions between flanking sequence regions and the polyQ tract. Specifically, the two flanking sequences reduce the favorable polyQ interaction surface and hence reduce the frequency of intermolecular interactions between and entanglements of the polyQ tract, thus slowing aggregation kinetics regardless of the molecular architecture at the monomer level. The latter affects the morphologies and number of aggregates that form during early aggregation events. Overall, we show that N17 and C38 diminish the formation of large, polydisperse early-stage aggregates that are intrinsic to polyQ. Our results also show how polyQ expansions can override the effects of flanking sequences. Taken together with cellular data, we propose that flanking sequences help diminish the formation of toxic species and this functionality is overwhelmed by mutational expansion of polyQ tracts. We use the conceptual framework of Janus colloids to propose a connection between our results and other aggregation prone systems because they can be partitioned into cores that drive aggregation and contexts that control the aggregation process.

Polyglutamine-rich suppressors of huntingtin toxicity act upstream of Hsp70 and Sti1 in spatial quality control of amyloid-like proteins.

Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q) toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS). High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic aggregation intermediates to the Hsp70/Sti1 system for spatial organization into benign species.

Role of Intrinsic Helicity Within N-Terminal Flanking Sequences on Huntingtin Aggregation Mechanisms

Huntington's disease (HD) is associated with CAG repeats within exon 1 that lead to polyglutamine (polyQ) expansions in the protein huntingtin (htt). Aberrant splicing of htt transcripts generates N-terminal fragments in a polyQ length dependent manner that span exon 1 of htt. These N-terminal fragments can be trafficked into the nucleus and form neuronal nuclear inclusions. Within exon 1 of htt the polyQ region is flanked by a N-terminal 17-residue amphipathic stretch (N17) and a C-terminal proline-rich stretch. Modulation of these flanking sequences has profound effects on HD progression. Specifically, post-translational modifications and single / double point mutations within N17 can modulate the intrinsic helicity of N17, as well as aggregation in vitro and HD phenotypes in animal models. However, the role of helicity within N17 on the modulation of polyQ aggregation remains unresolved. Here, we focus on understanding the synergy between the intrinsic helicity within N17 and polyQ length on htt aggregation mechanisms. Through the use of atomistic Monte Carlo simulations utilizing the ABSINTH implicit solvation model, we examine the monomeric properties of N17 permutants with varying degrees of intrinsic helicity as a function of polyQ length. Results suggest that the degree of coupling between N17 constructs and polyQ is dependent on the intrinsic helicity within N17 constructs. A comparative analysis of N17 constructs in cis with a range of polyQ lengths will be combined with biophysical experiments to understand how monomeric properties of N17 modulate the solubility and mechanism of polyQ aggregation.

Modulation of Polyglutamine Conformations and Associations by C-Terminal Proline Rich Regions from Exon 1 of Huntingtin

Huntington's disease (HD) is associated with CAG repeats within exon 1 that lead to polyglutamine (polyQ) expansions in the protein huntingtin (htt). Proteolysis of htt leads to products that can be trafficked into the nucleus and form neuronal intranuclear inclusions. The presence / absence of flanking sequences namely, the N-terminal 17-residue amphipathic stretch (Nt17h-htt) and the C-terminal 38-residue proline-rich stretch (Ct38h-htt) in products of proteolysis have a profound effect on the HD phenotype in animal models (where h-htt denotes human-htt). The biophysical basis for the effects of these flanking sequences remains unresolved. Here, we focus on the influence of different proline-rich C-terminal sequences on the conformations of and intermolecular associations through the polyQ tract. The flanking sequences include Ct38h-htt and Ct31m-htt (the mouse 31-residue C-terminal proline-rich region).We used atomistic simulations based on the ABSINTH implicit solvation model and underlying forcefield paradigm to quantify the nature of the coupling between C-terminal flanking sequences and polyQ tracts for different polyQ lengths. There are clear differences between the intrinsic and context dependent properties of Ct31m-htt and Ct38h-htt. Detailed analyses of the monomer conformations and intermolecular associations of polyQ in cis with different synthetic and naturally occurring C-terminal proline-rich stretches identify how flanking sequences modulate aggregation mechanisms. These results might be important for recent studies in animal models that show different toxicity profiles between transgenic animals expressing different C-terminal proline-rich sequences.This work was supported by grant 5R01NS056114 from the National Institutes of Health.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.

Solitary and Repetitive Binding Motifs for the AP2 Complex α-Appendage in Amphiphysin and Other Accessory Proteins

Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.

Mammalian Gene PEG10 Expresses Two Reading Frames by High Efficiency –1 Frameshifting in Embryonic-associated Tissues

Paternally expressed gene 10 (PEG10) is a mammalian gene that is essential for embryonic development in mice. The gene contains two overlapping open reading frames (ORF1 and ORF2) and is derived from a retroelement that acquired a cellular function. It is not known if both reading frames are required for PEG10 function. Synthesis of ORF2 would be possible only if programmed -1 frameshifting occurred during ORF1 translation. In this study the frameshifting activity of PEG10 was analyzed in vivo, and a potential role for ORF2 was investigated. Phylogenetic analysis demonstrated that PEG10 is highly conserved in therian mammals, with all species retaining the elements necessary for frameshifting as well as functional motifs in each ORF. The frameshift site of PEG10 was highly active in cultured cells and produced the ORF1-2 protein. In mice, endogenous ORF1 and an ORF1-2 frameshift protein were detected in the developing placenta and amniotic membrane from 9.5 days post-coitus through to term with a very high frameshift efficiency (>60%). Mutagenesis of the active site motif of a putative protease within ORF2 showed that this enzyme is active and participates in post-translational processing of PEG10 ORF1-2. Both PEG10 proteins were also detected in first trimester human placenta. By contrast, neither protein expression nor frameshifting was detected in adult mouse tissues. These studies imply that the ORF1-2 protein, synthesized utilizing the most efficient -1 frameshift mechanism yet documented in vivo, will have an essential function that is intrinsic to the importance of PEG10 in mammals.

Arrestin-2 Interacts with the Ubiquitin-Protein Isopeptide Ligase Atrophin-interacting Protein 4 and Mediates Endosomal Sorting of the Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4.arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.

A Novel Interaction between Atrophin-interacting Protein 4 and β-p21-activated Kinase-interactive Exchange Factor Is Mediated by an SH3 Domain

Cross-talk between G protein-coupled receptors and receptor tyrosine kinase signaling pathways is crucial to the efficient relay and integration of cellular information. Here we identify and define the novel binding interaction of the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) with the GTP exchange factor beta-p21-activated kinase-interactive exchange factor (beta PIX). We demonstrate that this interaction is mediated in part by the beta PIX-SH3 domain binding to a proline-rich stretch of AIP4. Analysis of the interaction by isothermal calorimetry is consistent with a heterotrimeric complex with one AIP4-derived peptide binding to two beta PIX-SH3 domains. We determined the crystal structure of the beta PIX-SH3.AIP4 complex to 2.0-A resolution. In contrast to the calorimetry results, the crystal structure shows a monomeric complex in which AIP4 peptide binds the beta PIX-SH3 domain as a canonical Class I ligand with an additional type II polyproline helix that makes extensive contacts with another face of beta PIX. Taken together, the novel interaction between AIP4 and beta PIX represents a new regulatory node for G protein-coupled receptor and receptor tyrosine kinase signal integration. Our structure of the beta PIX-SH3.AIP4 complex provides important insight into the mechanistic basis for beta PIX scaffolding of signaling components, especially those involved in cross-talk.

The role of the proline-rich domain of Ssdp1 in the modular architecture of the vertebrate head organizer.

Lim1, Ssdp1, and Ldb1 proteins are components of the Ldb1-associated transcriptional complex, which is important in the head-organizing activity during early mouse development. Depletion of each individual protein alone causes a headless phenotype. To explore in more detail the modular architecture of the complex, we have generated two different gene-trapped mouse lines that express truncated forms of Ssdp1. Embryos derived from the gene-trapped line that encodes a truncated Ssdp1 lacking the proline-rich sequence exhibit a lethal abnormal head-development phenotype, resembling mouse embryos deficient for Lim1, Ssdp1, or Otx2 genes. Embryos derived from the second gene-trapped line, in which most of the proline-rich domain of Ssdp1 is retained, did not show abnormalities in head development. Our data demonstrate that components of the Ldb1-dependent module can be subdivided further into discrete functional domains and that the proline-rich stretch of Ssdp1 is critical for embryonic head development. Furthermore, phylogenetic comparisons revealed that in Caenorhabditis elegans, a similar proline-rich sequence is absent in Ssdp but present in Ldb1. We conclude that although the overall architecture of the Ldb1-dependent module has been preserved, the genetic specification of its individual components has diversified during evolution, without compromising the function of the module.

PTPN11 (Shp2) Mutations in LEOPARD Syndrome Have Dominant Negative, Not Activating, Effects

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.

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.

“Natively Unfolded” VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity

Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus (deltaN70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the deltaN70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis- and trans-proteolytic activity of the deltaN70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta.

The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature.

Amphiphysin is a major dynamin-binding partner at the synapse; however, its function in fission is unclear. Incubation of large unilamellar liposomes with mice brain cytosol led to massive formation of small vesicles, whereas cytosol of amphiphysin 1 knockout mice was much less efficient in this reaction. Vesicle formation from large liposomes by purified dynamin was also strongly enhanced by amphiphysin. In the presence of liposomes, amphiphysin strongly affected dynamin GTPase activity and the recruitment of dynamin to the liposomes, but this activity was highly dependent on liposome size. Deletion from amphiphysin of its central proline-rich stretch dramatically potentiated its effect on dynamin, possibly by relieving an inhibitory intramolecular interaction. These results suggest a model in which maturation of endocytic pits correlates with the oligomerization of dynamin with either amphiphysin or other proteins with similar domain structure. Formation of these complexes is coupled to the activation of dynamin GTPase activity, thus explaining how deep invagination of the pit leads to fission.