Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.     

Characterization of a Cerebellar Granule Cell-Specific Gene Encoding the γ-Aminobutyric Acid Type A Receptor α6 Subunit

Abstract: The α6 subunit of γ-aminobutyric type A receptors is a marker for cerebellar granule cells and is an attractive candidate to study cell-specific gene expression in the brain. The mouse α6 subunit gene has nine exons and spans ～14 kb. The largest intron (intron 8) is ～7 kb. For a minority of mRNAs, a missplice of the first exon was identified that disrupts the signal peptide and most likely results in the production of nonfunctional protein. The gene is transcribed from a TATA-less promoter that uses multiple start sites. Using transgenic mice, it was found that the proximal 0.5 kb of the rat α6 gene upstream region confers expression on a β-galactosidase reporter gene. One founder gave rise to a line with cerebellar granule cell-specific expression, although expression varied with lobule region. Other founders had ectopic but neuron-specific expression, with β-galactosidase found in cerebellar Purkinje cells, neocortex, thalamus, hippocampus, caudate-putamen, and inferior colliculi. Thus, we have defined a region containing the basal promoter of the α6 subunit gene and that confers neuron-specific expression.     

The structure of the large regulatory α subunit of phosphorylase kinase examined by modeling and hydrogen‐deuterium exchange

Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)₄, and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full‐length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen‐deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N‐terminal glycoside hydrolase domain and a large C‐terminal importin α/β‐like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen‐deuterium exchange results obtained for this subunit as part of the (αβγδ)₄ PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter‐subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low‐exchanging region in the C‐terminus of α that is known to bind the regulatory domain of the catalytic γ subunit.     

Protein interactions with the platelet integrin αIIb regulatory motif

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin α‐subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet‐specific integrin α_IIbβ₃. We employ peptide‐affinity chromatography followed‐up with LC‐MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR‐peptide. Of these, seven have been reported in the PPI literature as interactors with integrin α‐subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin‐tagged α‐integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide‐affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Evidence for specific subunit distribution and interactions in the quaternary structure of α‐crystallin

The quaternary structure of α‐crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision‐induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of α‐crystallin. For total α‐crystallin isolated directly from fetal calf lens using size‐based chromatography, the αB‐crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the αA‐crystallin subunits. Furthermore, upon mixing molar equivalents of purified αA‐ and αB‐crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the αA‐ and αB‐crystallin homo‐oligomers was comparable, indicating that strength of the αA:αA, and αB:αB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of αB‐crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C‐terminus of αA‐crystallin was more susceptible to proteolytic attack in homo‐oligomers than that of αB‐crystallin. As subunit exchange proceeded, proteolysis of the αA‐crystallin C‐terminus increased, indicating that in the hetero‐oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the α‐crystallin domains and C‐terminal extensions of subunits within the α‐crystallin assembly. In particular, we propose that the palindromic IPI motif of αB‐crystallin gives rise to two orientations of the C‐terminus. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.     

Regulation of the rat interstitial collagenase promoter by IL-1β, c-Jun,and ras-dependent signaling in growth plate chondrocytes

In an attempt to better define molecular influences on rat interstitial collagenase gene expression in cartilage, the promoter function was characterized using transient transfection assay, electrophoresis mobility shift assay, and genetic analysis in isolated growth plate chondrocytes. Data from 5′-flanking deletion and selected mutations suggest that multiple cis elements in both the proximal and distal regions of the promoter were important in the regulation of promoter activity. A proximal tumor response element (TRE) was shown to be necessary for basal and interleukin (IL)-1β–inducible reporter gene activity. Cells stimulated by IL-1β (1 ng/ml; 18 h) had elevated TRE binding activity, and one of the factors involved was identified as the nuclear protein, c-Jun. Indeed, c-Jun directed antisense oligonucleotides reduced rat interstitial collagenase mRNA. A sense oligonucleotide was ineffective. Regulation of promoter activity was susceptible to Ras-dependent signaling as expression of dominant negative mutant of Ras kinase (pZIP-RasN17) reduced reporter gene activity. In a comparison of proximal promoter reporter plasmid activity between proliferative and hypertrophic cells, inhibition of Ras-dependent signaling was less effective in the later cell type. This study suggests that the activation of nuclear binding proteins that bind TRE may be a common event with IL-1β regulation. Moreover, these data suggest that the regulation of rat interstitial collagenase gene expression is a combinatorial process and multiple cis-acting regulatory sites may interact to exert different effects dependent on the stage of chondrocyte differentiation. J. Cell. Biochem. 67:92–102, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

Binding determinants of the small heat shock protein, αB‐crystallin: recognition of the ‘IxI’ motif

Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate‐prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB‐crystallin (‘αB’), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α‐crystallin domain (ACD) forms dimeric building blocks, while flanking N‐ and C‐termini direct the formation of larger sHSP oligomers. The most commonly observed inter‐subunit interaction involves a highly conserved C‐terminal ‘IxI/V’ motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB‐ACD. IxI‐mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers.     

cis elements that control the expression of chick aggrecan

Aggrecan is a large chondroitin sulfate proteoglycan whose expression is both cell-specific and developmentally regulated. Cloning and sequencing of the 1.8-kilobase genomic 5'-flanking sequence of the chick aggrecan gene revealed the presence of potential tissue-specific control elements including a consensus sequence found in the cartilage-associated silencers, CSIIS1 and CSIIS2, that were first characterized in the type II collagen promoter sequences, as well as numerous other cis elements. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively deleted portions of the chick aggrecan promoter and enhancer region demonstrated cell type-specific promoter activity and identified a 420-base pair region in the genomic 5-flanking region responsible for negative regulation of the aggrecan gene. In this report, three complementary methods, DNase I footprinting assays, transient transfections, and electrophoretic mobility shift assays (EMSA), provided an integral approach to better understand the regulation of the aggrecan gene. DNase I footprinting revealed that six regions of this genomic sequence bind to nuclear proteins in a tissue-specific manner. Transient transfection of reporter constructs bearing ablations of these protected sequences showed that four of the six protected sequences, which contain the sequence TCCTCC or TCCCCT, had repressor activities in transfected chick chondrocytes. Cross-competition EMSA using nuclear protein extracted from chondrocytes or fibroblasts explored the contributions of the different sequence elements in formation of DNA-protein complexes specific to cell type. This is the first parallel examination of the EMSA patterns for six functionally defined cis elements with highly similar sequences, using protein from primary cultured cells.     

EVIDENCE FOR A SPECIALIZED LOCALIZATION OF THE CHLOROPLAST ATP‐SYNTHASE SUBUNITS α, β, AND γ IN THE EYESPOT APPARATUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

The eyespot apparatus (EA) of Chlamydomonas reinhardtii P. A. Dang. consists of two layers of carotenoid‐rich lipid globules subtended by thylakoids. The outermost globule layer is additionally associated with the chloroplast envelope membranes and the plasma membrane. In a recent proteomic approach, we identified 202 proteins from isolated EAs of C. reinhardtii via at least two peptides, including, for example, structural components, signalling‐related proteins, and photosynthetic‐related membrane proteins. Here, we have analyzed the proteins of the EA with regard to their topological distribution using thermolysin to find out whether the arrangement of globules and membranes provides protection mechanisms for some of them. From about 230 protein spots separated on two‐dimensional gels, the majority were degraded by thermolysin. Five major protein spots were protected against the action of this protease. These proteins and some that were degradable were identified by mass spectrometry. Surprisingly, the thermolysin‐resistant proteins represented the α and β subunits of the soluble CF₁ complex of the chloroplast ATP synthase. Degradable proteins included typical membrane proteins like LHCs, demonstrating that thermolysin is not in general sterically prevented by the EA structure from reaching membrane‐associated proteins. A control experiment showed that the CF₁ complex of thylakoids is efficiently degraded by thermolysin. Blue native PAGE of thermolysin‐treated EAs followed by SDS‐PAGE revealed that the α and β subunits are present in conjunction with the γ subunit in a thermolysin‐resistant complex. These results provide strong evidence that a significant proportion of these ATP‐synthase subunits have a specialized localization and function within the EA of C. reinhardtii.