Fibronectin fibril formation involves cell interactions with two fibronectin domains

Fibronectin fragments and domain-specific antibodies have been used to study the mechanism by which cells reorganize exogenous fibronectin substrata into fibrils. Fibroblasts prevented from protein synthesis, and hence not secreting endogenous fibronectin or other matrix components, reorganized exogenous fibronectin substrata into arrays resembling the matrix of normally cultured cells. Cells also formed fibrils from substrata containing mixtures of cell- and either of two different heparin-binding fibronectin fragments but not from either fragment alone. The gelatin-binding fragment alone or in conjunction with the cell-binding fragment did not promote fibril formation. Antibodies recognizing cell- and either heparin- or the gelatin-binding domains labeled fibrils formed by cells under normal culture conditions or when a substratum of intact fibronectin was used as the sole exogenous source. However, only antibodies recognizing the cell- or either heparin-binding fragment reduced fibrillogenesis from intact fibronectin substrates when added during cell spreading. These data suggest that formation of fibronectin fibrils can occur at the cell surface and that membrane components recognizing the cell- and the heparin-binding domains in fibronectin may cooperate in the assembly process     

Structural domains of clathrin heavy chains

We used a combination of electron microscopy and proteolytic dissection to study the substructure of the clathrin trimer. The fragments of a heavy chain generated by limited proteolysis of cages were examined by rotary shadowing after disassembly. Correlation of lengths and molecular weights allowed us to map certain cleavage points along an arm and to assign them to positions in a model for a cage. We found that a particularly stable fragment of 52,000-59,000 Mr (depending on the enzyme) corresponded to the knob-like terminal domain at the tip of each arm.     

Evolution of distinct EGF domains with specific functions

EGF domains are extracellular protein modules cross-linked by three intradomain disulfides. Past studies suggest the existence of two types of EGF domain with three-disulfides, human EGF-like (hEGF) domains and complement C1r-like (cEGF) domains, but to date no functional information has been related to the two different types, and they are not differentiated in sequence or structure databases. We have developed new sequence patterns based on the different C-termini to search specifically for the two types of EGF domains in sequence databases. The exhibited sensitivity and specificity of the new pattern-based method represents a significant advancement over the currently available sequence detection techniques. We re-annotated EGF sequences in the latest release of Swiss-Prot looking for functional relationships that might correlate with EGF type. We show that important post-translational modifications of three-disulfide EGFs, including unusual forms of glycosylation and post-translational proteolytic processing, are dependent on EGF subtype. For example, EGF domains that are shed from the cell surface and mediate intercellular signaling are all hEGFs, as are all human EGF receptor family ligands. Additional experimental data suggest that functional specialization has accompanied subtype divergence. Based on our structural analysis of EGF domains with three-disulfide bonds and comparison to laminin and integrin-like EGF domains with an additional inter-domain disulfide, we propose that these hEGF and cEGF domains may have arisen from a four-disulfide ancestor by selective loss of different cysteine residues.     

Mapping two functional domains of clathrin light chains with monoclonal antibodies

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F. M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S. C. Harrison, P. Parham, and F. M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.     

Long, saturated chains: tasty domains for kinases of insulin resistance

The mechanistic basis of how cells respond to increased fatty acids (FAs) is murky but potentially involves receptor-mediated activation or inhibition by different FA classes. Holzer et?al. (2011) recently propose in Cell that expansion of intracellular membrane microdomains induced by saturated FA recruit and activate c-Src for JNK activation.     

Lectin activities of cytokines: functions and putative carbohydrate-recognition domains

The discovery that some cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain (CRD), which is localized at the opposite of the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity, which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase/phosphatase involved in receptor phosphorylation/dephosphorylation and in specific signal transduction. As a correlate, a cytokine can act only on cells possessing both the receptor and the ligand. Two cytokines using the same receptor can have different target cells and functions because of their different lectin activities. Based on a few examples, the CRD can be predicted based on the 3-D structures of the molecules.     

Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.     

Class-defining characteristics in the mouse heavy chains of variable domains

Analysis of residue correlation in over 2700 mouse heavy chains of the V(H) domains was carried out on three hierarchical levels. At the 'position' level, statistical analysis revealed 45 positions that conserve similar residues in almost all chains. At the 'fragment' level, the focus of investigation shifted to the study of combinations of amino acids in strands and loops. It was found that no more than 10 patterns were sufficient for describing strands and loops in the chains. At the 'sequence' level, we determined all possible combinations of these patterns and classified the mouse heavy chains. Comparison of the sequences in the eight classes revealed residues at the class-determining positions that were unique to each class. Because a strong correlation of residues was found, one only needs several residues to classify a sequence. It follows that no all residue alignment procedure is necessary to divide sequences into classes. An important corollary of our approach is the possibility of predicting residues in an incomplete sequence from a small sequence fragment. On the basis of our analysis of mouse heavy chains we hypothesize about the presently unknown mouse V(H) germline repertoire.     

Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions.

The three-dimensional structures of the copper-containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two-domain ancestor of the blue oxidases contained a trinuclear-copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper-containing, iron-regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.     

Characterization and comparative analyses of zebrafish intelectins: Highly conserved sequences,diversified structures and functions

Intelectin family, also called the X-lectin family, is a newly discovered gene family involved in development and innate immunity. However, no research was carried out for this gene family in the model organism zebrafish. Here we present the first characterization of seven zebrafish intelectins (zINTLs) and the first systematic comparative analysis of intelectins from various species in order to provide some clues to the function and evolution of this gene family. We examined the expression patterns of zINTLs in various development stages, normal adults, and Aeromonas salmonicida infected adults. Results showed that zINTL1–3 were highly expressed in one or several adult tissues. zINTL4–7, however, were expressed at quite low levels both in adults and various development stages, and some of them showed relaxation of functional constrains as revealed by K_a/K_s calculation. Of the seven zINTLs, zINTL3 was expressed predominantly in the liver and highly up-regulated upon infection, suggesting its important roles in immunity. Based on the characterization of zebrafish intelectins, we then conducted a systematic survey of intelectin members in various species and made comparative analyses. We found out that intelectin family may be a deuterostome specific gene family; and their expression patterns, quaternary structures and glycosylations vary considerably among various species, though their sequences are highly conserved. Moreover, these varied features have evolved multiple times independently in different species, resulting in species-specific protein structures and expression patterns.     

Functional protein domains from the thermally driven motion of polypeptide chains: A proposal

It is proposed that the thermally driven motion of certain polypeptide chains, including those that are part of an otherwise stable folded protein, produces time-averaged three-dimensional domains that confer unique functions to a protein. These domains may be controlled by collapsing the polypeptide into an enthalpically favored structure, or extending it into an entropically dominated form. In the extended form, these domains occupy a relatively large space, which may be used to regulate protein–protein interactions and confer mechanical properties to proteins. This “entropic bristle” model makes several predictions about the structure and properties of these domains, and the predictions are used to reevaluate a range of biophysical studies on proteins. The outcome of the analysis suggests that the entropic bristle can be used to explain a wide range of disparate and apparently unrelated experimental observations. Proteins 32:223–228, 1998. © 1998 Wiley-Liss, Inc.     

Multiple functions of polypeptides mediated by distinct domains interacting with different receptors

There exist multiple functions of polypeptide molecules. Both a polypeptide molecule interacting with one receptor, and distinct domains of the molecule interacting with different receptors could induce different intracellular signal transduction to elicit multiple functions. This review highlights the distinct domains of the polypeptide molecule interacting with different receptors to elicit multiple functions. It includes distinct domains, different receptor mechanisms, and different signal transduction of the polypeptide molecule.     

Transmembrane domains in the functions of Fc receptors

In the present study, we use a novel method, PHDhtm, to predict the exact locations and extents of the transmembrane (TM) domains of multisubunit immunoglobulin Fc-receptors. Whereas most previous studies have used single residue hydrophobicity plots for characterizing of these domains, PHDhtm utilizes a system of neural networks and the evolutionary information contained in multiple alignments of related sequences to predict the above. Present PHDhtm application predicts TM domains of immunoglobulin Fc-receptors that in many cases differ significantly from those derived by using earlier methods. Comparisons of helical wheel projections of the presently derived TM domains from PHDhtm with those produced earlier reveal different hydrophobic moments as well as hydrophobic and hydrophilic surfaces. These differences probably alter the character of subunit association within the receptor complexes. This new algorithm can also be used for other membrane protein complexes and may advance both understanding the principles underlying such complexes formation and design of peptides that can interfere with such TM domain association so as to modulate specific cellular responses.     

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)