Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases.

Angiogenesis underlies the majority of eye diseases that result in catastrophic loss of vision. Recent evidence has implicated the integrins alpha v beta 3 and alpha v beta 5 in the angiogenic process. We examined the expression of alpha v beta 3 and alpha v beta 5 in neovascular ocular tissue from patients with subretinal neovascularization from age-related macular degeneration or the presumed ocular histoplasmosis syndrome or retinal neovascularization from proliferative diabetic retinopathy (PDR). Only alpha v beta 3 was observed on blood vessels in ocular tissues with active neovascularization from patients with age-related macular degeneration or presumed ocular histoplasmosis, whereas both alpha v beta 3 and alpha v beta 5 were present on vascular cells in tissues from patients with PDR. Since we observed both integrins on vascular cells from tissues of patients with retinal neovascularization from PDR, we examined the effects of a systemically administered cyclic peptide antagonist of alpha v beta 3 and alpha v beta 5 on retinal angiogenesis in a murine model. This antagonist specifically blocked new blood vessel formation with no effect on established vessels. These results not only reinforce the concept that retinal and subretinal neovascular diseases are distinct pathological processes, but that antagonists of alpha v beta 3 and/or alpha v beta 5 may be effective in treating individuals with blinding eye disease associated with angiogenesis.

RXR gamma null mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/- mutant mice are viable.

The RXR gamma (RXR, retinoid X receptor) gene was disrupted in the mouse. Homozygous mutant mice developed normally and were indistinguishable from their RXR gamma +/- or wild-type littermates with respect to growth, fertility, viability, and apparent behavior in the animal facility. Moreover, RXR alpha -/-/RXR gamma -/- and RXR beta -/-/RXR gamma -/- mutant phenotypes were indistinguishable from those of RXR alpha -/- and RXR beta -/- mutants, respectively. Strikingly, RXR alpha +/-/RXR beta -/-/RXR gamma -/- triple mutants were viable. Thus, it appears that RXR gamma does not exert any essential function that cannot be performed by RXR alpha or RXR beta, and one copy of RXR alpha is sufficient to perform most of the functions of the RXRs.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence.

By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of karyopherin alpha. We found that the C-terminal region of karyopherin alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds karyopherin beta. Surprisingly, karyopherin alpha also contains an NLS. Thus, karyopherin alpha belongs to a group of proteins that contain both a ligand (NLS) and a cognate receptor (NLS recognition site) in one molecule with a potential for autologous ligand-receptor interactions. The NLS of karyopherin alpha overlaps with the binding site of karyopherin alpha for karyopherin beta. Hence, binding of karyopherin beta to karyopherin alpha covers the NLS of karyopherin alpha. This prevents autologous ligand receptor interactions and explains the observed cooperative binding of karyopherin alpha to a heterologous NLS protein in the presence of karyopherin beta.

Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

Function of the pre-T-cell receptor alpha chain in T-cell development and allelic exclusion at the T-cell receptor beta locus.

The pre-T-cell receptor, composed of the T-cell receptor (TCR) beta chain (TCRbeta), pre-Talpha (pTalpha) chain, and CD3 molecules, has been postulated to be a transducer of signals during the early stages of T-cell development. To examine the function of the transmembrane pTalpha chain during tbymocyte development, we generated pTalpha-/- embryonic stem cells and assayed their ability to differentiate into lymphoid cells in vivo after injection into recombination-activating gene (RAG)-2-deficient blastocysts. Thymocytes representing all stages of T-cell differentiation were detected in the thymus of pTalpha-/- chimeric mice, indicating that thymocyte development can occur without pTalpha. However, greatly reduced thymocyte numbers and substantially increased percentages of both CD4-CD8- thymocytes and TCRgammadelta+ thymocytes suggest that pTalpha plays a critical role in thymocyte expansion. To investigate the role of the pTalpha chain in allelic exclusion at the TCRbeta locus, a functionally rearranged TCRbeta minigene was introduced into pTalpha-/- and pTalpha+/- embryonic stem cells, which were subsequently assayed by RAG-2-deficient blastocyst complementation. In the absence of pTalpha, expression of the transgenic TCRbeta inhibited rearrangement of the endogenous TCRbeta locus to an extent similar to that seen in normal TCRbeta transgenic mice, suggesting that pTalpha may not be required for signaling allelic exclusion at the TCRbeta locus.

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, alpha L beta 2) integrin.

We report the 1.8-A crystal structure of the CD11a I-domain with bound manganese ion. The CD11a I-domain contains binding sites for intercellular adhesion molecules 1 and 3 and can exist in both low- and high-affinity states. The metal-bound form reported here is likely to represent a high-affinity state. The CD11a I-domain structure reveals a strained hydrophobic ridge adjacent to the bound metal ion that may serve as a ligand-binding surface and is likely to rearrange in the absence of bound metal ion. The CD11a I-domain is homologous to domains found in von Willebrand factor, and mapping of mutations found in types 2a and 2b von Willebrand disease onto this structure allows consideration of the molecular basis of these forms of the disease.

Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.

Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation.

In Alzheimer disease (AD) the amyloid beta-peptide (A beta) accumulates in plaques in the brain. A beta can be neurotoxic by a mechanism involving induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels ([Ca2+]i). In light of evidence for an inflammatory response in the brain in AD and reports of increased levels of tumor necrosis factor (TNF) in AD brain we tested the hypothesis that TNFs affect neuronal vulnerability to A beta. A beta-(25-35) and A beta-(1-40) induced neuronal degeneration in a concentration- and time-dependent manner. Pretreatment of cultures for 24 hr with TNF-beta or TNF-alpha resulted in significant attenuation of A beta-induced neuronal degeneration. Accumulation of peroxides induced in neurons by A beta was significantly attenuated in TNF-pretreated cultures, and TNFs protected neurons against iron toxicity, suggesting that TNFs induce antioxidant pathways. The [Ca2+]i response to glutamate (quantified by fura-2 imaging) was markedly potentiated in neurons exposed to A beta, and this action of A beta was suppressed in cultures pretreated with TNFs. Electrophoretic mobility-shift assays demonstrated an induction of a kappa beta-binding activity in hippocampal cells exposed to TNFs. Exposure of cultures to I kappa B (MAD3) antisense oligonucleotides, a manipulation designed to induce NF-kappa B, mimicked the protection by TNFs. These data suggest that TNFs protect hippocampal neurons against A beta toxicity by suppressing accumulation of ROS and Ca2+ and that kappa B-dependent transcription is sufficient to mediate these effects. A modulatory role for TNF in the neurodegenerative process in AD is proposed.

Reexpression of retinoic acid receptor (RAR) gamma or overexpression of RAR alpha or RAR beta in RAR gamma-null F9 cells reveals a partial functional redundancy between the three RAR types.

Disruption of retinoic acid receptor (RAR) gamma in F9 embryonal carcinoma cells leads to aberrent differentiation and reduced activation of expression of several all-trans-retinoic acid (RA)-induced genes. We have analyzed the expression of several additional RA-responsive genes in RAR alpha- and RAR gamma-null F9 cells. The RA-induced activation of Cdx1, Gap43, Stra4, and Stra6 was specifically impaired in RAR gamma-null cells, supporting the idea that each RAR may regulate distinct subsets of target genes. To further investigate the role of RAR gamma in F9 cell differentiation, "rescue" cell lines reexpressing RAR gamma 2 or overexpressing either RAR alpha 1 or RAR beta 2 were established in RAR gamma-null cells. Reexpression of RAR gamma or overexpression of RAR alpha restored both target-gene activation and the differentiation potential. In contrast, over-expression of RAR beta only poorly restored differentiation, although it could replace RAR gamma for the activation of target genes. Functional redundancy between the various RARs is discussed.

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells.

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.