Addition of electrophilic lipids to actin alters filament structure

Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and PGA(1) in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA(1) and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ(2) or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ(2) at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.     

GTP binding is required for SEPT12 to form filaments and to interact with SEPT11

Septins are a family of filament-forming GTP-binding proteins involved in a variety of cellular process such as cytokinesis, exocytosis, and membrane dynamics. Here we report the biochemical and immunocytochemical characterization of a recently identified mammalian septin, SEPT12. SEPT12 binds GTP in vitro, and a mutation (Gly56 to Asn) in the GTP-binding motif abolished binding. Immunocytochemical analysis revealed that wild-type SEPT12 formed filamentous structures when transiently expressed in Hela cells whereas SEPT12G56A generated large aggregates. In addition, wild-type SEPT12 failed to form filaments when coexpressed with SEPT12G56A. We also observed that GTP-binding by SEPT12 is required for interaction with SEPT11 but not with itself.     

Kar3 interaction with Cik1 alters motor structure and function

Kar3, a kinesin-14 motor of Saccharomyces cerevisiae required for mitosis and karyogamy, reportedly interacts with Cik1, a nonmotor protein, via its central, predicted coiled coil. Despite this, neither Kar3 nor Cik1 homodimers have been observed in vivo. Here we show that Kar3 is a dimer in vitro by analytical ultracentrifugation. The motor domains appear as paired particles by rotary-shadow electron microscopy (EM) and circular dichroism (CD) spectroscopy of the nonmotor region shows characteristics of helical structure, typical of coiled coils. Remarkably, the Kar3/Cik1 nonmotor region shows greater helicity by CD analysis and rotary-shadow EM reveals a stalk joined to one large or two smaller particles. The highly helical Kar3/Cik1 nonmotor region and visible stalk indicate that dimerization with Cik1 causes structural changes in Kar3. The Cik1 and Kar3 stalk regions preferentially associate with one another rather than forming homodimers. Kar3/Cik1 moves on microtubules at 2-2.4 microm min(-1), 2-5-fold faster than Kar3, and destabilizes microtubules at the lagging ends. Thus, structural changes in Kar3 upon dimerization with Cik1 alter the motor velocity and likely regulate Kar3 activity in vivo.     

Hematoporphyrin interacts with myoglobin and alters its functions

The binding parameters of hematoporphyrin, a photosensitizing drug used in photodynamic therapy, interacting with myoglobin, an oxygen storage protein, have been studied spectrofluorometrically and spectrophotometrically. Two concentration ranges of hematoporphyrin, representing significantly monomeric and aggregated (dimeric) states have been used. The binding affinity constant (K) decreases and the possible number of binding sites (p) increases as the porphyrin changes from significantly monomeric state to predominantly dimeric state. Titration of the protein with hematoporphyrin in a spectrophotometric study (differential spectroscopy) exhibits an isosbestic point indicating a ground state complex formation. The interaction leads to a conformational change of the protein as observed in a circular dichroism study. The hematoporphyrin-myoglobin interaction causes oxygen release from the protein and it varies with the stoichiometric ratio of the porphyrin:protein. Hematoporphyrin also increases the myoglobin-catalysed hydrogen peroxide-mediated oxidation of o-dianisidine and NADH. These findings on the effects of hematoporphrin-myoglobin interaction should be given due consideration in therapeutic uses of the porphyrin and its derivatives.     

Human spire interacts with the barbed end of the actin filament

Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell.     

The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.     

Nebulin interacts with CapZ and regulates thin filament architecture within the Z-disc

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant "molecular ruler" nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160-164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160-164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.     

The interaction of cofilin with the actin filament

Cofilin is a key actin-binding protein that is critical for controlling the assembly of actin within the cell. Here, we present the results of molecular docking and dynamics studies using a muscle actin filament and human cofilin I. Guided by extensive mutagenesis results and other biophysical and structural studies, we arrive at a model for cofilin bound to the actin filament. This predicted structure agrees very well with electron microscopy results for cofilin-decorated filaments, provides molecular insight into how the known F- and G-actin sites on cofilin interact with the filament, and also suggests new interaction sites that may play a role in cofilin binding. The resulting atomic-scale model also helps us understand the molecular function and regulation of cofilin and provides testable data for future experimental and simulation work.     

Immunoreactivity of the septins SEPT4, SEPT5, and SEPT8 in the human eye.

We aimed to examine the distribution of SEPT4, SEPT5, and SEPT8 in the human eye. For each septin, five to six normal human eyes were examined by immunohistochemical staining of paraffin sections using polyclonal antibodies against SEPT4, SEPT5, and SEPT8 and an avidin biotin complex immunodetection system. SEPT4 immunoreactivity (IR) was detected primarily in the epithelium of cornea, lens, and nonpigmented ciliary epithelium; in the endothelium of cornea and vessels of iris and retina; and in the retinal nerve fiber layer, the outer plexiform layer, the outer segments of the photoreceptor cells, the inner limiting membrane of the optic nerve head, and optic nerve axons. SEPT5-IR was present in corneal endothelial cells, iris tissue, nonpigmented ciliary epithelium, and epithelial cells of the lens. SEPT8-IR almost paralleled that of SEPT4, except for a lower SEPT8-IR of the outer photoreceptor segments and a positive staining of the meningothelial cell nests in the subarachnoidal space of the bulbar segment of the orbital optic nerve. In conclusion, SEPT4, SEPT5, and SEPT8 are expressed in various ocular tissues, each revealing a distinct expression pattern. Both physiological and potential pathophysiological role of septins in the human eye deserve further investigation.     

Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.     

Measles virus interacts with and alters signal transduction in T-cell lipid rafts

By a contact-dependent surface interaction, the measles virus (MV) glycoprotein complex induces a pronounced inhibition of T-cell proliferation. We now show that MV directly interacts with glycosphingolipid-enriched membrane microdomains on human primary T cells and alters recruitment and segregation of membrane proximal signaling components. Contact-dependent interference with T-cell receptor-stimulated tyrosine phosphorylation and Ca mobilization is a late event seen 24 h after MV treatment. In contrast, stimulated recruitment of pleckstrin homology domain-containing proteins such as Akt and Vav is inhibited early after MV contact, as is segregation of the activated Akt kinase from rafts. Tyrosine phosphorylation of the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), p85, is apparently normal then, yet this protein fails to partition to the lipid raft fraction, and this is associated with stable expression of its negative regulator Cbl-b. Thus, by interaction with lipid rafts, MV contact initially targets recruitment of PI3K by preventing stimulated Cbl-b degradation and activation of PI3K-dependent signaling components.     

Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cells

SHPS-1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with the protein tyrosine phosphatase SHP-2. Using a yeast two-hybrid screen, we identified a protein tyrosine kinase, Csk-homologous kinase (CHK), as an SHPS-1-interacting protein. Immunoprecipitation and pull-down assays using glutathione S -transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine-phosphorylated SHPS-1 via its SH2 domain. HIS3 assay in a yeast two-hybrid system using the tyrosine-to-phenylalanine mutants of SHPS-1 indicated that the first and second ITIMs of SHPS-1 are required to bind CHK. Over-expression of wild-type CHK, but not a kinase-inactive CHK mutant, enhanced the phosphorylation of SHPS-1 and its subsequent association with SHP-2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS-1 in vitro . Co-expression of SHPS-1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation.     

HDAC6 interacts with PTPN1 to enhance melanoma cells progression

Histone deacetylase 6 (HDAC6) plays an important role in oncogenic transformation and cancer metastasis. Our previous study has demonstrated that HDAC6 was highly expressed in melanoma cells, and contributed to the proliferation and metastasis of melanoma cells. However, the underlying mechanism of HDAC6 in melanoma metastasis and progression remains largely unclear. In this study, we reported that HDAC6 directly interacted with Tyrosine-protein phosphatase non-receptor type 1 (PTPN1) by performing co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). HDAC6 increased the protein level of PTPN1 independent of histone modifying activity. In addition, PTPN1 promoted proliferation, colony formation and migration while decreased apoptosis of melanoma cells through activating extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, we found that matrix metallopeptidase 9 (MMP9) was increased by HDAC6/PTPN1/ERK1/2 axis, which might serve as a mechanism for melanoma invasion and metastasis. In conclusion, HDAC6 might enhance aggressive melanoma cells progression via interacting with PTPN1, which was independent of its histone modifying activity.     

SCAMP2 interacts with Arf6 and phospholipase D1 and links their function to exocytotic fusion pore formation in PC12 cells

SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5'-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores.     

NUMBL interacts with TRAF6 and promotes the degradation of TRAF6

Tumor necrosis factor-associated factor 6 (TRAF6) is an essential adaptor protein for IL-1R or TLR-mediated NF-κB signaling pathway activation. In previous work we have found NUMBL interacts with TAB2 and negatively regulates NF-κB signaling pathway. Here, we report that NUMBL directly binds to TRAF6 in vivo and in vitro. NUMBL down-regulates TRAF6 protein level and shortens its half-life. Furthermore, knockdown of NUMBL significantly increases endogenous TRAF6 protein level in the cultured cortical neurons. In vivo ubiquitination assays indicate that NUMBL promotes the assembly of K48-linked polyubiquitination chains on TRAF6, but has no significant effect on its K63-linked polyubiquitination. Our results collectively reveal that NUMBL interacts with TRAF6 and promotes the degradation of TRAF6 in vivo, leading to the inhibition of NF-κB signaling pathway.     

Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.     

Acid-induced conformational modification of the hemagglutinin molecule alters interaction of influenza virus with antigen-presenting cells

Brief exposure of influenza virus to pH 5 was found to have extensive effects upon presentation of viral Th cell antigenic determinants. This acidity, comparable to that encountered in host cell endosomes, was known to effect conformational changes in the viral hemagglutinin (HA) which alter the molecule's fusion activity, antigenicity, and susceptibility to enzymes. Three major effects of low pH upon presentation of viral T cell determinants were observed: first, acid pretreatment permitted presentation by pre-fixed APC of two of three linear T cell sites of the HA molecule, bypassing the APC activity required to present untreated virus; second, the two determinants presented in this manner disappeared rapidly from APC surfaces; third, acid-pretreated virus was not efficiently utilized by active APC in the normal pathway of viral antigen presentation. These observations suggest that the pH-induced conformational transition of HA may constitute sufficient processing for certain linear determinants of the molecule and additionally influences the processes involved in the general formation and presentation of viral T cell sites.     

Disruption of the maxi-K-caveolin-1 interaction alters current expression in human myometrial cells

Background  

One determinant of the total K+ myometrial smooth muscle cell (MSMC) current is the large conductance, calcium- and voltage-activated potassium channel (maxi-K channel). This channel provides a repolarizing current in response to excitatory stimuli, most notably in response to increases in the levels of intracellular Ca2+, and blocking the channel by pharmacological means induces the depolarization of MSMCs and also enhances contraction strength. In MSMCs, maxi-K channels can reside in the caveolae, where they associate with the scaffolding protein caveolin-1 (cav-1). The aim of this study was to investigate the consequences of this interaction - more specifically, how disruption of the association between the maxi-K channel and cav-1 may influence the current expression and excitability of myometrial cells - with the aim of better understanding the mechanisms that underlie the regulation of normal and aberrant uterine function.     

Naloxone alters locomotion and interaction with environmental stimuli

Rats were injected with saline or naloxone (0.5, 5.0 or 25 mg/kg) and monitored for locomotion and both frequency and duration of contact with stimuli in a multicompartment exploratory chamber. Naloxone produced a dose-related reduction in locomotion and in frequency of contact with stimuli. At the lowest dose tested this reduction was accompanied by an increase in total duration of contact and in time spent per contact with the stimuli. In contrast, the highest dose of naloxone decreased the duration of contact with stimuli and induced prolonged periods of inactivity. An intermediate response was observed with 5.0 mg/kg naloxone. These results indicate that lower doses of naloxone may enhance interaction with environmental stimuli while the predominant effect of higher doses is a general suppression in behavioral activity. Similarities with the antimanic agent lithium chloride and possible clinical implications are discussed.