Structural requirements for thymosin beta4 in its contact with actin. An NMR-analysis of thymosin beta4 mutants in solution and correlation with their biological activity.

We examined the conformational preferences of mutants of thymosin beta4, an actin monomer sequestering protein by NMR spectroscopy in 60% (v/v) trifluoroethanol. Under these conditions, the wild-type thymosin beta4 conformation consists of an alpha-helix (helix I) extending from residues 5-16 with a more stable fragment from lysine 11 to lysine 16 and a second alpha-helix (helix II) encompassing residues 31-39. The point mutations studied here are located in helix I or in the LKKTET segment (residues 17-22) that form the two main entities of interaction with the actin molecule. The alpha-1H conformational shifts allow us to investigate the helicity of the polypeptides at the residue level and to correlate these structures with their biological activity. We determine that an extension of helix I at its C-terminal end over the LKK-segment results in loss of activity. The correct termination of this helix is connected to a specific orientation of the polypeptide essential for a cooperative action of the thymosin beta4 binding entities required for full activity.     

One-step procedure for the determination of thymosin beta 4 in small tissue samples and its separation from other thymosin beta 4-like peptides by high-pressure liquid chromatography

Thymosin beta 4 has been determined by a simple and fast one-step procedure in different tissues of rats. The tissues (1 to 40 mg) were disintegrated and deproteinized by homogenization in perchloric acid. After neutralization by potassium hydroxide the supernatant solution was used for determining thymosin beta 4 by reverse-phase HPLC without further manipulations. Not only does this procedure avoid artificial proteolysis as effectively as extraction of tissues by guanidinium chloride or boiling buffer, but it offers two further advantages. First, no additional steps--as for example desalting--are necessary prior to HPLC and thus the risk of losing thymosin beta 4 is eliminated. Using this procedure thymosin beta 4 is recovered quantitatively. The method is linear over the range 0.04 to 1.13 nmol and thymosin beta 4 is well separated from other thymosin beta 4-like peptides known to be present in mammals; i.e., thymosin beta Ala4, thymosin beta 9, thymosin beta 10, and thymosin beta Arg10. Second, the acid-insoluble pellet of the same extract can be used to determine the DNA content of the sample. Thus it is possible to relate thymosin beta 4 to DNA, which then allows comparing cells of different tissues and cell lines to one another. This procedure is also applicable to small peptides soluble in perchloric acid.     

Simultaneous isolation and determination of prothymosin alpha, parathymosin alpha, thymosin beta 4, and thymosin beta 10

A method was described for the isolation of peptides from rat thymus. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from rat thymus were, in micrograms per gram of whole tissue, 60-80 for prothymosin alpha, 50-80 for thymosin beta 4, and 20-30 for thymosin beta 10. The procedure also yielded smaller quantities of a fourth peptide, designated parathymosin alpha. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin alpha and thymosin beta 4 to the boiled tissue extract.     

Cell-cycle-regulated expression of thymosin beta 4 in thymocytes.

Thymosin beta 4 belongs to a family of ubiquitous peptides present at a high cellular content but still with an unknown intracellular function. The expression of this peptide was studied in concanavalin-A-stimulated, proliferating rat thymocytes during cell cycle progression. An early, transient 10-fold increase of the peptide occurred 1 h after stimulation without elevation of the corresponding mRNA level. This increase coincided with that of thymosin beta 4 biosynthesis. The sharp decline of the thymosin beta 4 content was not due to a secretion of the peptide into the medium. During S phase and mitosis, the biosynthetic rates as well as mRNA content, but not the cellular thymosin beta 4 concentration, increased again. After 96 h of culture the values returned to those of quiescent cells.     

A micro-scale method for determining relative metal-binding affinities of proteins

This article describes a quick and easy method for determining relative binding affinities between proteins and metal ions. The method is based on separating unbound metal ions from metal ions bound to protein by ultrafiltration using microcentrifuge ultrafiltration units. Bovine serum albumin (BSA) was used as the test protein and the relative affinity towards divalent metal ions was found to be Cu²⁺>Zn²⁺>Cd²⁺>Pb²⁺>Ni²⁺>Co²⁺, which corresponds to the relative orders reported in the literature.     

A method for detection of phage mutants with altered transducing ability

Summary A method is described which allows the detection of phage mutants with increased or decreased transduction frequencies. Some properties of such mutants are presented.     

Amber mutants in gene 67 of phage T4. Effects on formation and shape determination of the head

Two amber mutations in gene 67 of bacteriophage T4 were constructed by oligonucleotide-directed mutagenesis and the resulting mutated genes were recombined back into the phage genome and their phenotype was studied. The 67amK1 mutation is close to the amino terminus of the gene, and phage carrying this mutation are unable to form plaques on suppressor-negative hosts. A second mutation, 67amK2, which lies in the middle of the gene, three codons N-terminal to a proteolytic cleavage site, produces a small number of viable phage particles. In suppressor-negative hosts, both mutants produce polyheads and proheads. 67amK1 assembles only few proheads that have a disorganized core structure, as judged from thin sections of infected cells. The proheads and the mature phages of both mutants are mainly isometric rather than having the usual prolate shape. Depending on the 67 mutant and the host, between 20% and 73% of the particles that are produced are isometric, and 1 to 10% are two-tailed biprolate particles. 67amK2 phages grown on a supD suppressor strain that inserts serine in place of the wild-type leucine do not contain gp67* derived from gene product 67 (gp67) by proteolytic cleavage. This demonstrates the importance of the correct amino acid at this position in the protein. Other abnormalities in these 67amK2 phages are the presence of uncleaved scaffolding core proteins (IPIII and gp68), indicating a structural alteration in the prohead scaffold, resulting in only partial cleavage. In wild-type phages these proteins are found in the head only in the cleaved form. With double-mutants of 67 with mutations in the major shell protein gp23 no naked scaffolding cores were found, confirming the necessity of gp67 for the assembly or persistence of a "normal" core.     

Folding stability modulation of the villin headpiece helical subdomain by 4‐fluorophenylalanine and 4‐methylphenylalanine

HP36, the helical subdomain of villin headpiece, contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, and Phe58). Hydrophobic effects and electrostatic interactions were shown to be the critical factors in stabilizing this core and the global structure. To assess the interactions among Phe47, Phe51, and Phe58 residues and investigate how they affect the folding stability, we implanted 4‐fluorophenylalanine (Z) and 4‐methylphenylalanine (X) into the hydrophobic core of HP36. We chemically synthesized HP36 and its seven variants including four single mutants whose Phe51 or Phe58 was replaced with Z or X, and three double mutants whose Phe51 and Phe58 were both substituted. Circular dichroism and nuclear magnetic resonance measurements show that the variants exhibit a native HP36 like fold, of which F51Z and three double mutants are more stable than the wild type. Molecular modeling provided detailed interaction energy within the phenylalanine residues, revealing that electrostatic interactions dominate the stability modulation upon the introduction of 4‐fluorophenylalanine and 4‐methylphenylalanine. Our results show that these two non‐natural amino acids can successfully tune the interactions in a relatively compact hydrophobic core and the folding stability without inducing dramatic steric effects. Such an approach may be applied to other folded motifs or proteins. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 627–637, 2015.     

A decoy set for the thermostable subdomain from chicken villin headpiece,comparison of different free energy estimators

Background  

Estimators of free energies are routinely used to judge the quality of protein structural models. As these estimators still present inaccuracies, they are frequently evaluated by discriminating native or native-like conformations from large ensembles of so-called decoy structures.     

Expression pattern of thymosin beta 4 in the adult human liver

Thymosin beta-4 (Tβ4) is a member of beta-thymosins, a family of small peptides involved in polymerization of G-actin, and in many critical biological processes including apoptosis, cell migration, angiogenesis, and fibrosis. Previous studies in the newborn liver did not reveal any significant reactivity for Tβ4 during the intrauterine life. The aim of the present study was to investigate by immunohistochemistry Tβ4 expression in the adult normal liver. Thirty-five human liver samples, including 11 needle liver biopsies and 24 liver specimens obtained at autopsy, in which no pathological change was detected at the histological examination, were immunostained utilizing an anti-Tβ4 commercial antibody. Tβ4 was detected in the hepatocytes of all adult normal livers examined. A zonation of Tβ4 expression was evident in the vast majority of cases. Immunostaining was preferentially detected in zone 3, while a minor degree of reactivity was detected in periportal hepatocytes (zone 1). At higher power, Tβ4-reactive granules appeared mainly localized at the biliary pole of hepatocytes. In cases with a strong immunostaining, even perinuclear areas and the sinusoidal pole of hepatocytes appeared interested by immunoreactivity for Tβ4. The current work first evidences a strong diffuse expression of Tβ4 in the adult human liver, and adds hepatocytes to the list of human cells able to synthesize large amounts of Tβ4 in adulthood. Moreover, Tβ4 should be added to the liver proteins characterized by a zonate expression pattern, in a descending gradient from the terminal vein to the periportal areas of the liver acinus. Identifying the intimate role played by this peptide intracellularly and extracellularly, in physiology and in different liver diseases, is a major challenge for future research focusing on Tβ4.     

A method for the isolation of phage mutants altered in their response to lysogenic induction

Summary Phage cl ⁺ gives clear plaques whereas phage cIind ^- gives turbid plaques on a lawn of a mutant strain of E. coli K12. This strain, called STS, carries mutation spr in a tif sfi genetic background. I hypothesize that upon temperate phage infection, STS bacteria spontaneously inactivate phage repressor by the same mechanism involved in normal lysogenic induction which results in obligatory lytic growth of ⁺. The use of the STS mutant facilitates the isolation and genetic analysis of phage mutants with an abnormal response to lysogenic induction.     

Biosynthesis rates and content of thymosin beta 4 in cell lines

The content and relative biosynthetic rates of thymosin beta 4 have been determined in 28 different cell lines. The highest content of thymosin beta 4 as well as the highest rate of biosynthesis was observed in Epstein-Barr virus-transformed human B-cell lines. The levels observed in these cells are 1 pg thymosin beta 4 per cell, which is three times higher than that in rat peritoneal macrophages. Thus, these B-cell lines have the highest content of thymosin beta 4 of any cell type yet described. Since all of the Epstein-Barr virus-transformed B-cells described here grow in suspension, it is unlikely that the presence of thymosin beta 4 is related to anchorage in these cells. Thymosin beta 4 is not secreted by viable Epstein-Barr virus-transformed B cells in culture, suggesting some intracellular function of the peptide. These results indicate that these B-cell lines may be suitable for the study of thymosin beta 4 function.     

Immunoreactivity of thymosin beta 4 in human foetal and adult genitourinary tract

Thymosins beta 4 (Tβ4) is a member of the beta-thymosins family, a family of peptides playing essential roles in many cellular functions. Our recent studies suggested Tβ4 plays a key role in the development of human salivary glands and the gastrointestinal tract. The aim of this study was to analyse the presence of Tβ4 in the human adult and foetal genitourinary tract. Immunolocalization of Tβ4 was studied in autoptic samples of kidney, bladder, uterus, ovary, testicle and prostate obtained from four human foetuses and four adults. Presence of the peptide was observed in cells of different origin: in surface epithelium, in gland epithelial cells and in the interstitial cells. Tβ4 was mainly found in adult and foetal bladder in the transitional epithelial cells; in the adult endometrium, glands and stromal cells were immunoreactive for the peptide; Tβ4 was mainly localized in the glands of foetal prostate while, in the adults a weak Tβ4 reactivity was restricted to the stroma. In adult and foetal kidney, Tβ4 reactivity was restricted to ducts and tubules with completely spared glomeruli; a weak positivity was observed in adult and foetal oocytes; immunoreactivity was mainly localized in the interstitial cells of foetal and adult testis. In this study, we confirm that Tβ4 could play a relevant role during human development, even in the genitourinary tract, and reveal that immunoreactivity for this peptide may change during postnatal and adult life.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.     

The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.     

Interaction of thymosin beta 4 with muscle and platelet actin: implications for actin sequestration in resting platelets.

Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.