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S Huang

Publications and source records attributed to S Huang.

At least 343 records · Page 19Linked to original sources
PubMed1997

Memory of extracellular adenosine A2A purinergic receptor-mediated signaling in murine T cells.

Accumulation of extracellular and intracellular adenosine (Ado) under hypoxic conditions or in the absence of adenosine deaminase results in lymphocyte depletion and in severe combined immunodeficiency, which are currently explained by direct intracellular lymphotoxicity of Ado metabolites. In support of the alternative, "signaling" mechanism, we show that extracellular Ado (extAdo) suppresses all tested T cell receptor (TCR)-triggered effector functions of T lymphocytes including the TCR-triggered FasL mRNA up-regulation in cytotoxic T lymphocytes. Strong evidence against the intracellular lymphotoxicity of Ado (and in support of the signaling model) is provided by abrogation of TCR-triggered growth inhibition in Ado-exposed T cells. The brief exposure to Ado was sufficient to observe inhibition of TCR-triggered effector functions. The "memory" of T cells to exposure to extAdo is best explained by sustained increases in cAMP. Selective agonist (CGS21680) and antagonist (ZM241385) of A2A adenosine receptor were used in functional assays and cDNA probes for different sybtypes of adenosine receptors were used in Northern blot studies. A2A receptors are identified as the predominantly expressed subtype of Gs-coupled Ado receptors in T cells. The demonstration of cross-talk between the A2A receptors and TCR in both directions support the possible role of A2A receptors in mechanisms of extAdo-mediated immunosuppression in vivo under adenosine deaminase deficiency and hypoxic conditions in, e.g., solid tumors.

Adenosine↗
PubMed1997

Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion.

Accumulation of adenosine and of deoxyadenosine in the absence of adenosine deaminase activity (ADA) activity results in lymphocyte depletion and in severe combined immunodeficiency (ADA SCID), which is currently explained by direct cell death-causing effects of intracellular products of adenosine metabolism. We explored the alternative mechanisms of peripheral T-cell depletion as due to inhibition of T-cell expansion by extracellular adenosine-mediated signaling through purinergic receptors. The strong inhibition of the T-cell receptor (TCR)-triggered proliferation and of upregulation of interleukin-2 receptor alpha chain (CD25) molecules, but not the direct lymphotoxicity, were observed at low concentrations of extracellular adenosine. These effects of extracellular adenosine (Ado) are likely to be mediated by A2a receptor-mediated signaling rather than by intracellular toxicity of adenosine catabolites, because (1) poorly metabolized adenosine analogs cause the accumulation of cAMP and strong inhibition of TCR-triggered CD25 upregulation; (2) the A2a, but not the A1 or A3, receptors are the major expressed and functionally coupled adenosine receptors in mouse peripheral T and B lymphocytes, and the adenosine-induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; (3) the specific agonist of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, whereas the specific antagonist of A2a receptor, CSC, inhibits the effects of Ado and CGS21680; and (4) the increases in [cAMP]i mimic the adenosine-induced inhibition of TCR-triggered CD25 upregulation and splenocyte proliferation. These studies suggest the possible role of adenosine receptors in the regulation of lymphocyte expansion and point to the downregulation of A2a purinergic receptors on T cells as a potentially attractive pharmacologic target.

Adenosine↗
PubMed1997

Nitric oxide-mediated apoptosis of K-1735 melanoma cells is associated with downregulation of Bcl-2.

Recent studies have shown that the treatment of nonmetastatic K-1735 murine melanoma cells with cytokines induces the production of nitric oxide (NO) and hence cell death. The purpose of this study was to determine the mechanism of this cytokine-induced NO-mediated apoptosis. Incubation of nonmetastatic K-1735 cells with interleukin-1 alpha (IL-1alpha) and interferon-gamma (IFN-gamma) induced high NO production, Bcl-2 downregulation, and apoptotic cell death. In contrast, incubation of metastatic K-1735 cells with IL-1alpha and IFN-gamma did not induce significant production of NO, downregulation of Bcl-2, or cell death. The exposure to exogenous NO derived from the NO donors, sodium nitroprusside (SNP), or GEA5024 produced a dose-dependent apoptotic cell death in both the metastatic and nonmetastatic K-1735 cells, which was associated with downregulation of Bcl-2 at the mRNA level and, to a lesser extent, at the protein level. Nonmetastatic and metastatic K-1735 cells transfected with the Bcl-2 gene were more resistant to apoptosis mediated by both endogenous and exogenous NO. Subsequent to intravenous injection, the tumor cells transfected with the Bcl-2 gene had an increased survival rate in the lungs of nude mice and produced a higher number of experimental lung metastases. These data suggest that NO-induced apoptosis in K-1735 melanoma cells is associated with downregulation of Bcl-2.

Animals↗
PubMed1997

Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes.

Epstein-Barr virus (EBV) infection is associated with several human diseases that involve unrestricted proliferation of B lymphocytes. EBV nuclear antigen 1 (EBNA-1) is expressed in all EBV-infected cells and plays an essential role in persistence of the EBV genome. EBNA-1 has also been reported to have oncogenic potential. As an approach for treating EBV infections, we examined the capacity of EBNA-1 ribozymes delivered by recombinant adenoviruses to suppress EBNA-1 expression and to block virus-induced B cell proliferation. In contrast to primary B cells, EBV-transformed B lymphoblastoid cell lines expressed alphav integrins, the adenovirus internalization receptors, and were also susceptible to adenovirus-mediated gene delivery. Adenovirus delivery of a specific ribozyme (RZ1) to lymphoblastoid cell lines, suppressed EBNA-1 mRNA and protein expression, significantly reduced the number of EBV genomes, and nearly abolished cell proliferation in low serum. Adenovirus delivery of RZ1 also prevented EBV infection of an established EBV-negative B cell line. These studies demonstrate the potential use of adenovirus-encoded ribozymes to treat EBV-induced lymphoproliferative disorders.

Adenoviridae↗
PubMed1997

Expression, assembly, and proteolytic processing of Helminthosporium victoriae 190S totivirus capsid protein in insect cells.

The dsRNA genome (5.2 kbp) of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs). The 5' proximal ORF codes for the capsid protein (CP) and the 3' ORF codes for an RNA-dependent RNA polymerase. Although the capsid of Hv190SV is encoded by a single gene, it is composed of two major closely related polypeptides, either p88 and p83 or p88 and p78. Whereas p88 and p83 are phosphoproteins, p78 is nonphosphorylated. Expression of the CP ORF in insect cells generated both p78 and p88 which assembled into virus-like particles. The finding that p78, p83, and p88 share a common N-terminal amino acid sequence is consistent with the determination that N-terminal, but not C-terminal, CP deletions were incompetent for assembly. Evidence was obtained that p78 is derived from p88 via proteolytic cleavage at the C-terminus. Proteolytic processing may play a regulatory role in the virus life cycle since it leads to dephosphorylation of CP and a subsequent decrease in virion transcriptional activity.

Animals↗
PubMed1997

Cytosolic domain of the type I interleukin-1 receptor spontaneously recruits signaling molecules to activate a proinflammatory gene.

Immediate postreceptor events activated by IL-1-IL-1R interaction remain undefined. We have initiated studies to identify candidate signal transducers that associate with the cytosolic domain (cd) of the IL-1R. Immunocomplex kinase assays demonstrated an IL-1-activated myelin basic protein kinase activity that coprecipitated with the IL-1R from rat mesangial, mouse EL-4, and HeLa cells. Using glutathione-S-transferase (GST) fusion proteins, HeLa cell lysates next were assayed for kinases that associated with IL-1R cytoplasmic sequences. A GST-IL-1R fusion protein containing the entire cd (amino acids 369-569; GST-IL-1Rcd) recruited a kinase activity in the absence and presence of IL-1 stimulation. In contrast, a GST-IL-1R membrane-proximal region mutant (amino acids 369-501; GST-IL-1RcdDelta), which lacks COOH-terminal amino acid residues required for nuclear factor-kappaB activation, poorly phosphorylated MBP. In gel, kinase assays demonstrated 63-, 83-, and 100-kD kinases that specifically coprecipitated with the HeLa IL-1R and the GST-IL-1Rcd, but not GST-IL-1RcdDelta. 35S-labeled proteins, with Mrs identical to the kinase activities, stably associated with GST-IL-1Rcd. Transient transfection assays of 293 cells were used to evaluate the functional significance of these findings. Simply increasing IL-1cd expression in 293 cells stimulated 5'-IL-6 flanking region-regulated CAT activity threefold above control, an effect blocked by the kinase inhibitors staurosporine and calphostin C. In summary, we have identified two previously unrecognized 63- and 83-kD kinases as well as a protein with an Mr similar to the recently cloned IL-1R-associated kinase, all of which associate spontaneously with the IL-1Rcd. Ectopic IL-1Rcd expression was sufficient to trigger cellular activation, suggesting that the extracellular domain of the intact receptor represses signal transduction until IL-1 is bound. Given that the IL-1Rcd signaling domain has been conserved in a functionally diverse group of transmembrane receptors, further characterization of this signaling process may define novel molecular mechanisms controlling cellular function and differentiation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

The dynamic organization of the perinucleolar compartment in the cell nucleus.

The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P H1 RNA) and the polypyrimidine tract binding protein (PTB; hnRNP I) have thus far been identified in the PNC (Ghetti, A., S. PinolRoma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181-1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471-11476). In this report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human cancer and normal cells showed that PNCs are much more prevalent in cancer cells. Analysis through the cell cycle using immunolabeling with a monoclonal antibody, SH54, specifically recognizing PTB, demonstrated that the PNC dissociates at the beginning of mitosis and reforms at late telophase in the daughter nuclei. To visualize the PNC in living cells, a fusion protein between PTB and green fluorescent protein (GFP) was generated. Time lapse studies revealed that the size and shape of the PNC is dynamic over time. In addition, electron microscopic examination in optimally fixed cells revealed that the PNC is composed of multiple strands, each measuring approximately 80-180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Furthermore, analysis of the sequence requirement for targeting PTB to the PNC using a series of deletion mutants of the GFP-PTB fusion protein showed that at least three RRMs at either the COOH or NH2 terminus are required for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.

Adenocarcinoma↗
PubMed1997

High susceptibility to collagen-induced arthritis in mice lacking IFN-gamma receptors.

Collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis, is induced in DBA/1 (H-2q) mice following immunization with type II collagen (CII) in CFA. Since we have previously shown that IFN-gamma exerts a biphasic effect during the evolution of CIA in DBA/1 mice, we analyzed the development of this disease in mice with a disruption of the IFN-gamma receptor gene (IFN-gammaR(0/0)). Mutant mice were interbred with the DBA/1 strain to yield IFN-gammaR(0/0) mice expressing the H-2q haplotype. In three consecutive experiments, IFN-gammaR(0/0) male mice were found to exhibit severe clinical and histologic arthritis with an average incidence of 88.5 vs 94.1% for the wild DBA/1 strain. Notably, onset of clinical symptoms occurred significantly earlier than in DBA/1 mice. Although of a lower magnitude than in males, CIA also developed early in IFN-gammaR(0/0) female mice and with higher clinical severity than in control DBA/1 females. Immunization of knockout mice with CII resulted in the generation of CII-specific T cells belonging to the Th1 phenotype that recognize the same immunodominant peptides as do DBA/1 mice. CIA in IFN-gammaR(0/0) mice was associated with a down-regulation of the CII-specific IgG response, and this impairment was essentially due to a strong reduction of Abs of the IgG2a isotype. Taken together, our findings provide evidence that IFN-gammaR deficiency in DBA/1 mice leads to the occurrence of severe CIA with an accelerated onset compared with that in wild-type mice, indicating that the proinflammatory action of IFN-gamma has been bypassed in the IFN-gammaR(0/0) mice.

Animals↗
PubMed1997

Accelerated collagen-induced arthritis in IFN-gamma receptor-deficient mice.

Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis. Here, we describe experiments showing that IFN-gamma receptor knockout (IFN-gammaR alpha KO) mice of the DBA/1 strain develop CIA more readily than their wild-type counterparts. Symptoms of disease started 10 days earlier and the cumulative incidence of arthritis was significantly higher in the mutant mice than in wild-type mice. Similarly, accelerated onset of the disease was also found in wild-type DBA/1 mice treated with neutralizing mAbs against IFN-gamma. Histologic examination of the joints revealed a massive infiltration of the synovium with mononuclear cells and neutrophils, hyperplasia, and severe pannus formation in IFN-gammaR alpha KO mice when such inflammatory lesions were not yet detectable in wild-type mice. Serum levels of anti-collagen type II Abs, including total IgG and IgM, as well as IgG1, IgG2a, and IgG2b isotypes were found to be lower in the mutant mice. IL-2 and IL-4 remained undetectable in sera of both groups of mice, but did appear in the circulation after anti-CD3 Ab challenge. Significantly higher IL-2 and lower IL-4 serum levels were found in anti-CD3-challenged IFN-gammaR alpha KO mice than in wild-type counterparts, both at an early and at a later stage of the disease. These observations indicate that endogenous IFN-gamma counteracts development of collagen-induced arthritis and suggest that IFN-gamma does so by up-regulating IL-4 production and/or down-regulating IL-2 production. The data are in line with the concept of a pathogenic role of Th1-type cellular immunity in CIA in spite of a decreased Ab response to collagen type II.

Animals↗
PubMed1997

Expression of MCAM/MUC18 by human melanoma cells leads to increased tumor growth and metastasis.

The cell surface adhesion molecule MCAM (MUC18) is strongly expressed by advanced primary and metastatic melanomas but is weaker and less frequent in nevus cells. Previous studies have shown that MCAM expression correlates with tumor thickness and metastatic potential of human melanoma cells in nude mice. To provide direct evidence that MCAM plays a role in tumor growth and metastasis of human melanoma, the nonmetastatic MCAM-negative primary cutaneous melanoma SB-2 cells were transfected with MCAM cDNA and analyzed subsequently for changes in their tumorigenic and metastatic potential. Enforced expression of MCAM in SB-2 cells rendered them highly tumorigenic and increased their metastatic potential in nude mice as compared with parental and control transfected cells. The transfected cells displayed increased homotypic adhesion, increased attachment to human endothelial cells, decreased ability to adhere to laminin, and increased invasiveness through Matrigel-coated filters. Anti-MCAM monoclonal antibody reversed these functions in the transfected cells but not in control cells. The above changes in function attributed to the expression of MCAM may underlie the contribution of MCAM/MUC18 to the malignant phenotype.

Animals↗
PubMed1997

Geometric control of cell life and death.

Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment.

Animals↗
PubMed1997

Quaternary structure sensitive tyrosine interactions in hemoglobin: a UV resonance Raman study of the double mutant rHb (beta99Asp-->Asn, alpha42Tyr-->Asp).

Two interactions involving tyrosines have been implicated in the communication pathway that links ligand binding to quaternary state changes in hemoglobin. Tyr alpha(1)42 stabilizes the alpha1beta2 T state interface through the formation of a hydrogen bond to Asp beta(2)99. The side chains of the penultimate Tyr residues (alpha140 and beta145) occupy the pockets made by helicies F and H in the deoxy form with the phenolic hydroxyl hydrogen bonded to the carbonyl group of Val FG5. Early crystallographic studies indicated that in the R form the penultimate Tyr is expelled out of the pocket, thus eliminating the hydrogen bond. This hydrogen bond has been considered to play an important role in maintaining the low-oxygen-affinity state (T state) in deoxy HbA, but a later higher resolution crystallographic study (Shannon, 1983) failed to reveal such movement of this Tyr during the R --> T transition. Nevertheless, conversion of this Tyr to Phe increases oxygen affinity considerably, suggesting that hydrogen bonding is involved in oxygen affinity modulation. Earlier ultraviolet resonance Raman results reported by Spiro and co-workers [Rodgers et al. (1992) J. Am. Chem. Soc. 114, 3697-3709] were used to conclude that the significant quaternary structure dependent changes observed in tyrosine Raman bands are due to the formation of the T state hydrogen bond with Tyr alpha42 acting as a proton acceptor rather than being the anticipated proton donor, as would be expected if Asp beta99 were ionized. This surprising result rests on the assumption that changes in the environment of Tyr alpha42 are the overwhelming contributor to the R - T UV Raman difference spectrum. In this study, a cooperative double mutant lacking Tyr alpha42, [rHb (Asp beta99 --> Asn, Tyr alpha42 --> Asp)], is used to determine the relative contributions of Tyr alpha42 and the penultimate tyrosines to the R - T UV resonance Raman difference spectrum. The results both directly support the claim that Tyr alpha42 is the proton acceptor in the T state and expose the potential role of the penultimate tyrosines in coupling the quaternary state to the ligand reactivity.

Allosteric Regulation↗
PubMed1997

Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene.

BACKGROUND: The activation of an enzyme, inducible nitric oxide synthase (iNOS), catalyzes the production of endogenous nitric oxide (NO). NO, in turn, is associated with cell death, suppression of tumor development, and inhibition of metastasis of murine melanoma cells. Moreover, the in vivo induction of iNOS is associated with regression of established hepatic metastases. Whether this regression required the activation of the iNOS gene in every tumor cell or whether NO-producing tumor cells can also kill bystander (neighboring) cells has been previously unknown. PURPOSE: The goal of this study was to determine whether cells producing NO and then undergoing autolysis can also kill bystander cells in vitro and in vivo. METHODS: Murine K-1735 C4.parental (C4.P) melanoma cells were transfected with the functional iNOS gene (transfectant denoted as C4.L8) or with a control truncated-nonfunctional iNOS gene (transfectant denoted as C4.S2). NO-mediated cytolysis and bystander cell killing were determined in vitro and in vivo. RESULTS: Only the functional iNOS-transfected C4.L8 cells produced NO and underwent autolysis. C4.L8 cells also produced statistically significant lysis of iNOS-negative C4.P cells. This lysis was suppressed by the specific iNOS inhibitor N(G)-methyl-L-arginine. NO-producing C4.L8 cells and control C4.P or C4.S2 cells were injected subcutaneously into syngeneic C3H/HeN mice. Control C4.P and C4.S2 cells produced rapidly growing subcutaneous tumors, whereas C4.L8 cells did not. The mixture of C4.P and C4.S2 cells (1:5 ratio) produced rapidly growing subcutaneous tumors, whereas the mixture of C4.P and C4.L8.5 cells (1:5 ratio) produced slow-growing tumors. The subcutaneous growth of C4.P cells was not affected by C4.L8.5 cells injected subcutaneously at a distant site. Mixtures of C4.P cells labeled with [(125)I]iododeoxyuridine and C4.L8 cells (NO producing) or C4.S2 cells (control) were injected subcutaneously. The survival rate of the radiolabeled cells indicated that the NO-producing C4.L8.5 cells lysed the bystander C4.P cells. CONCLUSION: The production of high-level endogenous NO causes autolysis in tumor cells and lysis of bystander cells under in vitro and in vivo conditions. IMPLICATIONS: NO-mediated cell killing does not require transfection of genes into every cell in a neoplasm.

Animals↗
PubMed1997

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization.

Interaction of the adenovirus penton base protein with alpha v integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg-Gly-Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo-electron microscopy (cryo-EM) and image reconstruction using a mAb (DAV-1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV-1 binding corresponded to the weak density above each of the five 22 A protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus-Fab cryo-EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies.

Adenoviridae↗
PubMed1997

Human papillomavirus types 52 and 58 are prevalent in cervical cancers from Chinese women.

A substantial body of evidence has confirmed human papillomavirus (HPV) infection as an etiologic agent in human cervical cancer. To evaluate the association between HPV and cervical cancer in Chinese women, we examined tumor specimens from women who lived in Shanghai, People's Republic of China. Biopsies from 40 women, diagnosed with either squamous-cell carcinoma (n = 35) or adenocarcinoma (n = 5) were tested for HPV DNA by PCR. The HPV types present in tumors were determined either by hybridization of PCR products with HPV type-specific probes or by PCR-based sequencing. A total of 35 of the 40 cervical cancer specimens (87.5%) contained HPV DNA. The following distribution and types were detected: 7.5% HPV 16, 10% HPV 18, 20% HPVs 16 and 18, 15% HPV 52, 15% HPV 58, 12.5% HPVs 52 and 58 and 7.5% unclassified HPVs. In this population of Chinese women with cervical cancer, HPV 52 and 58 were as prevalent as the "high-risk" (for cervical cancer) viruses HPVs 16 and 18.

Adenocarcinoma↗
PubMed1997

The retinoblastoma interacting zinc finger gene RIZ produces a PR domain-lacking product through an internal promoter.

The PR domain is a newly recognized protein motif that characterizes a subfamily of Krüppel-like zinc finger genes. Members of the PR domain family have been shown to play important roles in cell differentiation and malignant transformation. The RIZ gene is the founding member of this family; it was isolated because its gene products can bind to the retinoblastoma tumor suppressor protein. Here, we have studied the RIZ gene structure and expression. By immunoprecipitation and immunoblot analysis we identified two different RIZ protein products of 280 and 250 kDa, designated RIZ1 and RIZ2, respectively. The 280-kDa RIZ1 product comigrated with the RIZ cDNA-derived polypeptide. The 250-kDa RIZ2 product lacked the NH2-terminal PR domain of RIZ1; it comigrated with a truncated RIZ1 polypeptide that was initiated from an internal ATG codon. Both the full-length and the truncated RIZ1 polypeptide were located in the nucleus as shown by transfection and immunofluorescence analysis. We identified the RIZ2 transcripts and showed that they were produced by an internal promoter located at the 5' boundary of coding exon 5. RNase protection analysis revealed similar ratios of RIZ1 and RIZ2 transcripts in most adult rat tissues except in testis, where RIZ1 was more abundant than RIZ2. These observations were strikingly similar to those described for the MDS1-EVI1 cancer gene, which also normally gives rise to a PR domain-lacking product, EVI1, because of an internal promoter.

Amino Acid Sequence↗
PubMed1997

Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex.

Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin. Dissociation of the tight profilactin complex may serve as a unique mechanism by which profilin helps regulate actin filament growth.

Actins↗