Yuzhu Zhang

Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments

ABSTRACT HEF1, p130Cas, and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115HEF1, p105HEF1, p65HEF1, and p55HEF1. We show that p115HEF1 and p105HEF1 are different phosphorylation states of the full-length HEF1. p55HEF1, however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105HEF1 and p115HEF1 being rapidly upregulated upon induction of cell growth, whereas p55HEF1 is produced specifically at mitosis. While p105HEF1 and p115HEF1 are predominantly cytoplasmic and localize to focal adhesions, p55HEF1 unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G2/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55HEF1. In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.

Evidence that dim1 associates with proteins involved in pre-mRNA splicing, and delineation of residues essential for dim1 interactions with hnRNP F and Npw38/PQBP-1.

The small evolutionarily conserved protein Dim1p/hDim1/Dib1p/DML-1 was initially defined as a factor essential for progression through the G2/M transition, and shown to be required to maintain the steady state level of a component of the fission yeast anaphase promoting complex/cyclosome. More recently, Dib1p has been defined as a component of the U4/U6.U5 tri-snRNP, required for pre-mRNA splicing. To investigate the mechanism(s) of Dim1 function, reiterative two-hybrid screening was performed to identify interacting proteins. Proteins thus identified were solely those involved in pre-mRNA splicing or related functions, and one partner induced a striking synthetic phenotype when co-expressed with hDim1 in mammalian cells. Saturating alanine scanning mutagenesis of Dim1 allowed delineation of amino acids essential for its ability to interact with its defined partners: mapping these residues on the structural coordinates of hDim1 defined an interactive sector of the protein. Finally, depletion studies have recently shown that Dim1 function is essential for pre-mRNA splicing in yeast. We find that elimination of DML-1 expression in C. elegans by RNA interference leads to embryonal lethality during gastrulation, marked by a failure to correctly express early zygotic transcripts. These results parallel the arrest phenotypes associated with global disruption of zygotic gene expression, suggesting that Dim1 proteins maintain an essential function in gene expression in higher eukaryotes.

Crystal structure of Ara h 3, a major allergen in peanut.

The prevalence of food allergy has increased dramatically in recent years. Tremendous research progress has been made in understanding the pathophysiological mechanisms of allergy and in identifying and characterizing food allergens. Peanut is a major food allergen source and Ara h 3 is a major peanut allergen. Using overlapping short peptides, several linear IgE-binding epitopes in Ara h 3 have been defined before. However, the structure of Ara h 3 of the native allergen is not clear and information on conformational epitopes is lacking. Structural characterization of allergens is required for understanding the allergenicity of food allergens and for the development of immunotherapeutic agents. Previously, we have reported the crystallization of Ara h 3 purified from raw peanut. Here we report the crystal structure of Ara h 3 at 1.73A resolution. Mapping of the previously defined linear epitopes on the crystal structure of Ara h 3 indicated that linear epitopes with more solvent exposure were those indicated by the literature to react with more patient sera. The structure of Ara h 3 may be used to assess the importance of conformational epitopes in further investigations.

Crystal structure of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

Seed storage proteins are accumulated during seed development and act as a reserve of nutrition for seed germination and young sprout growth. Plant seeds play an important role in human nutrition by providing a relatively inexpensive source of protein. However, many plant foods contain allergenic proteins, and the number of people suffering from food allergies has increased rapidly in recent years. The 11S globulins are the most widespread seed storage proteins, present in monocotyledonous and dicotyledonous seeds as well as in gymnosperms (conifers) and other spermatophytes. This family of proteins accounts for a number of known major food allergens. They are of interest to both the public and industry due to food safety concerns. Because of the interests in the structural basis of the allergenicity of food allergens, we sought to determine the crystal structure of Pru1, the major component of the 11 S storage protein from almonds. The structure was refined to 2.4 A, and the R/Rfree for the final refined structure is 17.2/22.9. Pru1 is a hexamer made of two trimers. Most of the back-to-back trimer-trimer association was contributed by monomer-monomer interactions. An alpha helix (helix 6) at the C-terminal end of the acidic domain of one of the interacting monomers lies at the cleft of the two protomers. The residues in this helix correspond to a flexible region in the peanut allergen Ara h 3 that encompasses a previously defined linear IgE epitope.

Purification, crystallization and preliminary X-ray characterization of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

Characterization of catechin-α-lactalbumin conjugates and the improvement in β-carotene retention in an oil-in-water nanoemulsion.

The goal of this study was to prepare and characterize α-lactalbumin (ALA)-catechin conjugates as a novel emulsifier in improving the retention of β-carotene (BC) in nanoemulsions via a free radical method. Covalent modification was observed and at least one catechin molecule was binding with ALA according to ESI-MS results. Far-UV CD indicated that the secondary structure of ALA was changed after conjugation. The Z-average particle diameters of nanoemulsions stabilized with ALA and ALA-catechin conjugates were 158.8 and 162.7 nm, respectively. The increase of mean particle size and the degradation of BC at 50°C were both larger than at 25°C during 30 days storage. BC retention stabilized with ALA-catechin conjugates was appreciably greater than ALA (control), which was attributed to the increase of ALAs radicals-scavenging and free metal ion binding ability after grafting with catechin. The chemical antioxidant activities of ALA-catechin conjugates were increased with increasing concentrations from 0.1 to 1.0mg/ml. In general, labile phytochemicals, like BC, can be protected against oxidation during storage by proteins-polyphenols conjugates without any side effects.

Characterization of milk proteins–lutein complexes and the impact on lutein chemical stability

In this study, the interaction of WPI (whey protein isolate) and SC (sodium caseinate) with hydrophobic lutein was investigated through UV-vis spectroscopy and circular dichroism (CD) as well as fluorescence. The effects on luteins chemical stability were also examined. The decrease of turbidity of lutein suggested that luteins aqueous solubility was improved after binding with milk proteins. CD analysis indicated lutein had little impact on the secondary structures of both proteins. Different preparation methods have significant impacts on the binding constant. Fluorescence results indicated that WPI and SC interact with lutein by hydrophobic contacts. Milk proteins have protective effects on lutein against oxidation and decomposition, and SC showed better capability in protecting lutein from oxidation than WPI during 16 days storage. The luteins chemical stability was increased with increasing of proteins concentration. The results indicated that milk proteins may act as effective carriers for lipophilic nutraceuticals.

Crystal Structure of Peanut (Arachis hypogaea) Allergen Ara h 5

Profilins from numerous species are known to be allergens, including food allergens, such as peanut ( Arachis hypogaea ) allergen Ara h 5, and pollen allergens, such as birch allergen Bet v 2. Patients with pollen allergy can also cross-react to peanut. Structural characterization of allergens will allow a better understanding of the allergenicity of food allergens and their cross-reactivities. The three-dimensional structures of most known food allergens remain to be elucidated. Here, we report the first crystallographic study of a food allergen in the profilin family. The structure of peanut allergen Ara h 5 was determined, and the resolution of the final refined structure was 1.1 Å. Structure alignment revealed that Ara h 5 is more similar to Bet v 2 than to Hev b 8, although sequence alignment suggested that Ara h 5 is more closely related to Hev b 8 than to Bet v 2, indicating that homology-model-based prediction of immunoglobulin E epitopes needs to be interpreted with caution.

Purification and characterization of the 7S vicilin from Korean pine (Pinus koraiensis).

Pine nuts are economically important as a source of human food. They are also of medical importance because numerous pine nut allergy cases have been recently reported. However, little is known about the proteins in pine nuts. The purpose of this study was to purify and characterize pine nut storage proteins. Reported here is the first detailed purification protocol of the 7S vicilin-type globulin from Korean pine (Pinus koraiensis) by gel filtration, anion exchange, and hydrophobic interaction chromatography. Reducing SDS-PAGE analysis indicated that purified vicilin consists of four major bands, reminiscent of post-translational protease cleavage of storage proteins during protein body packing in other species. The N-terminal ends of vicilin peptides were sequenced by Edman degradation. Circular dichroism (CD) and differential scanning calorimetry (DSC) analyses revealed that pine nut vicilin is stable up to 80 degrees C and its folding-unfolding equilibrium monitored by intrinsic fluorescence can be interpreted in terms of a two-state model.

A 1.55 A resolution X-ray crystal structure of HEF2/ERH and insights into its transcriptional and cell-cycle interaction networks.

Functional complementation screens can identify known or novel proteins with important intracellular activities. We have isolated human enhancer of filamentation 2 (HEF2) in a screen to find human genes that promote pseudohyphal growth in budding yeast. HEF2 is identical to enhancer of rudimentary homolog (ERH), a highly conserved protein of 104 amino acids. In silico protein‐interaction mapping implies that HEF2/ERH interacts with transcription factors, cell‐cycle regulators, and other proteins shown to enhance filamentous growth in S. cerevisiae, suggesting a context for studies of HEF2/ERH function. To provide a mechanistic basis to study of HEF2/ERH, we have determined the crystal structure of HEF2/ERH at 1.55 Å. The crystal asymmetric unit contains a HEF2/ERH monomer. The two monomers of the physiological dimer are related by the y, x, –z crystal symmetric operation. The HEF2/ERH structure is characterized by a novel α + β fold, a four‐strand antiparallel β‐sheet with three α‐helixes on one side of the sheet. The β‐sheets from the two monomers together constitute a pseudo‐β‐barrel, and form the center of the functional HEF2/ERH dimer, with a cavity channel at the dimer interface. Docking of this structure to the HEF2/ERH partner protein DCOH/PCD suggests that HEF2/ERH may regulate the oligomeric state of this protein. These data suggest that HEF2/ERH may be an important transcription regulator that also functions in the control of cell‐cycle progression. Proteins 2007.