Expression of α4 Integrin mRNA and Protein and Fibronectin in the Early Chicken Embryo

α₄ integrins (α₄β₁ and α₄β₇) have been shown to mediate both cell-matrix adhesion to fibronectin and cell-cell adhesion to VCAM-1. These interactions have been suggested to contribute to hematopoiesis, lymphocyte homing, recruitment of inflammatory cells, neural crest cell migration and myogenesis. We report here the cloning of chicken α₄ cDNA and its use to define the patterns of expression of α₄ mRNA and protein in early chicken embryos (19–22 somite pairs), a stage at which neural crest cells can be examined at various points in their migration and somitic development and differentiation can also be observed at various stages. We observe widespread expression of both α₄ mRNA and protein, although the patterns of steady state expression do not conform precisely. Many neural crest cells contain significant levels of α₄ mRNA. Some neural crest cells express α₄ protein but its expression is transient and/or limited to a subset of these cells. α₄ is strongly expressed at both mRNA and protein levels by somitic cells and their derivatives in the sclerotome, dermatome and myotome and is also expressed in neural tube, otic placode, heart, gut endoderm and some other tissues. Comparison with the distributions of fibronectin shows that, although some α₄ expression occurs in locations consistent with a role in cell-matrix adhesion to fibronectin, α₄ is also expressed in other places where fibronectin is low or absent and a role for α₄ in cell-cell interactions appears more likely.     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

Chromatin Protein HP1α Interacts with the Mitotic Regulator Borealin Protein and Specifies the Centromere Localization of the Chromosomal Passenger Complex

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1α (HP1α) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1α. This borealin-HP1α interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1α interaction, demonstrating the spatial requirement of HP1α in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1α and CPC localization in the centromere and illustrate the critical role of borealin-HP1α interaction in orchestrating an accurate cell division.     

Interleukin-1β Increased the Expression of Protease-Activated Receptor 4 mRNA and Protein in Dorsal Root Ganglion Neurons

Protease-activated receptor-4 (PAR4) is localized in primary sensory neurons and is believed to implicate in the modulation of nociceptive mechanisms. The pro-inflammatory cytokine interleukin-1β (IL-1β) is involved in the generation of hyperalgesia in pathological states such as neuropathy and inflammation. Previous studies have shown that IL-1β enhances the expression of PAR4 in many cell types but the effect of this cytokine on primary sensory neuron PAR4 expression is less clear. In the present study, we evaluated in rat dorsal root ganglion (DRG) neurons the influence of IL-1β on PAR4 mRNA and protein levels after IL-1β intraplantar injection into the hind-paw or treatment of cultured DRG neurons. The expression of PAR4 in cultured DRG neurons was also assessed after treatment with IL-1β with pre-addition of phorbol-12-myristate 13-acetate (PMA, a PKC activator) or chelerythrine chloride (a PKC inhibitor). We found that IL-1β intraplantar injection into the hind-paw or long-term exposure of cultured DRG neurons to IL-1β significantly increased the proportion of DRG neurons expressing PAR4 immunoreactivity. Real-time PCR and western blotting showed that IL-1β treatment also significantly elevated PAR4 mRNA and protein levels in DRG neurons. This IL-1β effect was enhanced in DRG neurons when DRG cultures were pre-treatment with the PMA. But pre-incubation with chelerythrine chloride strongly inhibited the IL-1β-induced increase of PAR4 mRNA and protein levels. These results demonstrate that the expression of PAR4 mRNA and protein induced by IL-1β is PKC signaling pathway dependent.     

Characterization of the interaction between Actinin-Associated LIM Protein (ALP) and the rod domain of α-actinin

Background  

The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, α-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown.     

Protein aggregation and amyloidosis: confusion of the kinds?

Recent years have witnessed major advances in our understanding of the structural basis of protein aggregation on several fronts. Firstly, high-resolution structural information that remained elusive for many years was provided by a series of studies of amyloid fibers using NMR, X-ray crystallography and electron microscopy, thereby confirming earlier models based on lower resolution observations. Secondly, studies of the sequence determinants of protein aggregation culminated in the development of computer algorithms that predict aggregation-prone sequences with good accuracy, allowing the design of mutations that reduce aggregation. Thirdly, based on the first results from such predictions and on statistical analysis of naturally occurring aggregating sequences, a picture is emerging in which aggregation-prone sequences are capped by gatekeeper residues that oppose aggregation. In addition to their aggregation-opposing function, it seems that gatekeeper residues are also important in determining chaperone selectivity for strongly aggregating regions. Finally, recent computational and experimental work shows that preventing aggregation does not necessarily mean that amyloid formation is prevented and vice versa. Thus, although aggregation and amyloidosis correlate to a certain extent, they are different processes and should be treated as such.     

Biochemical Systematics of Gymnosperms (4)—Seed Protein Peptides and Needle Peroxidases of Taxodiaceae

An analysis of seed protein peptides by using SDS polyacrylamide gel electrophoresis, indicates that all members in Taxodiaxeae share three peptides with molecular weights of 24, 32 and 10 kilodaltons. Generally, seeds of this family contain 2 to 4 major peptides, and their molecular weights are distributed in two regions: 24-26 K and 29-36 K respectively. The quantity of these major peptides is near or even more than 50% of seed total protein content. This kind of protein profiles is similar to that of Taxaceae and Cephalotaxaceae rather than to that of Pinaceae. On the other hand, a considerable difference was observed in peptides with higher molecular weights. Among different species within a genus, no difference has been found both in their seed protein profiles and in their needle peroxidases. Based on the data cited here, it seems that the relationships among most genera of Taxodiaceae are not so closed as the rela-tionship among following genera: Sequoia, Sequoiadendron and Taxodium.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex,Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPPα secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the γ-secretase complex, resulting in a reduction of the PS1 active fragment blocking Aβ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active γ-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of Aβ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the γ-secretase and affect the APP processing pathway.     

Nuclease Activity of the Human SAMHD1 Protein Implicated in the Aicardi-Goutières Syndrome and HIV-1 Restriction

The human HD domain protein SAMHD1 is implicated in the Aicardi-Goutières autoimmune syndrome and in the restriction of HIV-1 replication in myeloid cells. Recently, this protein has been shown to possess dNTP triphosphatase activity, which is proposed to inhibit HIV-1 replication and the autoimmune response by hydrolyzing cellular dNTPs. Here, we show that the purified full-length human SAMHD1 protein also possesses metal-dependent 3′→5′ exonuclease activity against single-stranded DNAs and RNAs in vitro. In double-stranded substrates, this protein preferentially cleaved 3′-overhangs and RNA in blunt-ended DNA/RNA duplexes. Full-length SAMHD1 also exhibited strong DNA and RNA binding to substrates with complex secondary structures. Both nuclease and dNTP triphosphatase activities of SAMHD1 are associated with its HD domain, but the SAM domain is required for maximal activity and nucleic acid binding. The nuclease activity of SAMHD1 could represent an additional mechanism contributing to HIV-1 restriction and suppression of the autoimmune response through direct cleavage of viral and endogenous nucleic acids. In addition, we demonstrated the presence of dGTP triphosphohydrolase and nuclease activities in several microbial HD domain proteins, suggesting that these proteins might contribute to antiviral defense in prokaryotes.     

The Role of Various Domains of the Iron–Sulfur Protein in the Assembly and Activity of the Cytochromme bc 1 Complex of Yeast Mitochondria

Assembly studies in vitro of deletion mutants of the iron–sulfur protein into the cytochromebc ₁ complex revealed that mutants localized in the extramembranous regions of the proteinwere not assembled into the complex in contrast to the efficient assembly of mutants in themembrane-spanning region. Charged amino acids located in the extramembranous 1-4 loopand the 1 helix were mutated and expressed in yeast cells lacking the gene for the iron–sulfurprotein. Mutating the charged amino acid residues H124, E125, R146, K148, and D149 aswell as V132 and W152 resulted in loss of enzymatic activity due to the loss of iron–sulfurprotein suggesting that these amino acids are required to maintain protein stability. By contrast,no loss of iron–sulfur protein accompanied the 30–50% loss of bc ₁ complex activity in mutantsof three conserved alanine residues, A86, A90, and A92, suggesting that these residues maybe involved in the proposed movement of the flexible tether of the iron–sulfur proteinduring catalysis.     

Sorting and Processing of the Alzheimer’s Disease Amyloid Precursor Protein Mediated by the AP-4 Complex

Proteolytic processing of the transmembrane amyloid precursor protein (APP) to aggregation-prone amyloid-β (Aβ) peptide underlies the development of Alzheimer’s disease.     

Allosteric Regulation of the Human and Mouse Deoxyribonucleotide Triphosphohydrolase Sterile α-Motif/Histidine-Aspartate Domain-containing Protein 1 (SAMHD1)

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G₁, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3–4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent K_m for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower K_m constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.