Protein Z4 Research Articles

The zinc-finger protein Z4 cooperates with condensin II to regulate somatic chromosome pairing and 3D chromatin organization.

Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.

The impact of terroir on barley and malt quality – a critical review

Why was the work done: With respect to terroir, ‘To be or not to be,’ (Hamlet, Shakespeare) is a key question for maltsters and brewers for malt and beer quality. Terroir is a sparsely studied aspect of malt quality, despite it being an important component of added market-value in wine since ancient times. The ‘sense of place’ imbued by terroir is an expression of the growth of a grape variety in a specific region with respect to local climate, soil, microbiome, elevation/aspect of the vineyard, viticultural and wine making methods. Similar corollaries to wine terroir can be drawn for hops and malting barley. How was the work done: A comprehensive review of the literature was undertaken to identify reports of terroir in barley/malt quality. Where possible, the discussion was extended by consideration of appropriate unpublished data. What are the main findings: The primary influence of terroir on malt is grain protein content (GPC). This appears, in part, to be controlled by the daylength during grain maturation. Increasing day length typical of grain maturation in Australia tends to be associated with a lower GPC, while decreasing daylength during grain maturation in Canada is associated with a higher protein content. GPC is positively correlated with diastatic power, beta-amylase activity and foam positive proteins such as protein Z4 and hordeins. Conversely, GPC is inversely correlated with extract and Kolbach Index (KI). Interestingly, lower protein barley tends to produce higher KI malts that result in wort with greater flavour complexity and desirability. The level of ionic micronutrients (cations, anions) including calcium and zinc are understudied aspects of malt quality. It is evident that there is significant variation in the ionic micronutrient content of malt produced from different international regions and between regions of the same country which would be an expression of terroir. Lastly, the microbiome of barley/malt shows influences of terroir such as the deleterious impacts of Fusarium head blight on malt quality including gushing and mycotoxins. Variation in terroir will also have more subtle impacts, both desirable and undesirable, on malt quality for the contribution of beneficial enzymes (e.g., cell wall degrading enzymes) or for the propensity of barley to impart malt components into wort (e.g., arabinoxylan) that have been implicated in premature yeast flocculation (PYF) and undesirable beer quality. Why is the work important: The concept of terroir in malt quality has important implications for the efficiency of mashing, lautering, fermentation and beer quality.

Phenotypic and allelic variation for wort protein Z in Australian and Canadian barleys

Protein Z is a major component in beer foam. Two-dimensional electrophoresis was used to analyze wort proteins of two Australian (Buloke and Commander) and two Canadian (CDC Meredith and Bentley) varieties. The Canadian barley contained more abundant proteins from MW 40–45 kDa (pI 5 to 7). These proteins were identified as either protein Z4 or protein Z7 using liquid chromatography–mass spectrometry. Full-length gene of protein Z4 and Z7 were sequenced from Canadian and Australian barleys. Sequence differences were identified in the coding region and upstream regions of the two genes, resulting in protein sequence and expression variations. Molecular markers were designed according to the indels in the upstream regions of protein Z4 and Z7 genes. These markers were highly correlated to wort protein Z content in Canadian and Australian varieties. The Canadian barleys contained ‘high level’ genotypes for protein Z4 and Z7 while most Australian barleys had ‘low level’ genotypes for protein Z4, Z7 or both. The markers identified in this study provide a valuable tool for the selection of protein Z alleles in marker-assisted breeding. Total protein Z content was assessed using different steeping conditions, and increasing air-rest time increased protein Z content in 15 varieties.

ITRAQ-based quantitative proteomic analysis of key barley proteins reveals changes after malting

Since the choice of malting varieties, as well as the impact of malting on protein composition and other malt quality parameters, are fundamental for beer production, there was a need to focus on this study. The present work was undertaken to evaluate the effect of malting processing on the barley proteins. The relationship between the protein pattern of barley grains and malt was investigated by combination of HPLC SEC separation with MS. We have found that the biggest difference between grain and malt protein profiles was observed in the fraction, where these proteins were identified: β-amylase, β-glucosidase and two various isoforms of protein Z (Z4, Z7). This peak was significantly increased after the malting. In addition, iTRAQ was applied for investigation of the relative quantitative changes of selected proteins after food technological processing. Our results indicate that the content of β-amylase is increased more rapidly in comparison to β-glucosidase. On the other hand, protein Z7 seems to have the opposite trend. The quantitative analysis reveals the increasing tendency of protein Z4 and decreasing tendency of Z7 protein after malting.

Comparative analysis of the effect of protein Z4 from barley malt and recombinant Pichia pastoris on beer foam stability: Role of N-glycosylation and glycation

This study aimed to elaborate the effect of N-glycosylation and glycation of protein Z4 from barley malt and recombinant Pichia pastoris on beer foam stability. The malt protein Z4 and recombinant protein Z4 showed similar N-glycosylation patterns while recombinant protein Z4 was glycosylated at a higher degree. In the simulated mashing and boiling, malt protein Z4 and deglycosylated malt protein Z4 preferred to glycate with glucose and maltose while recombinant protein Z4 and deglycosylated recombinant protein Z4 showed preference towards fructose. The addition of protein Z4 and protein Z4-saccharide complexes in finished beer showed that the addition of glycosylated protein Z4 only slightly enhanced the beer foam stability while the addition of glycated protein Z4 and protein Z4 with both glycation and glycosylation could significantly increase the beer foam stability. Therefore, glycation instead of N-glycosylation of protein Z4 played important roles in maintaining beer foam stability.

Proteome-Based Analysis of Colloidal Instability Enables the Detection of Haze-Active Proteins in Beer.

Colloidal haze is a serious quality defect of bright beers that considerably reduces their shelf life and is thought to be triggered by hordeins, a class of proline-rich barley proteins. In this work, the proteomes of fresh and old beers were investigated in bottled pilsners and compared to the protein inventory of haze to identify specific haze-active proteins. Haze isolates dissolved in rehydration buffer contained high concentrations of proteins and sugars but provided protein gels with weak spot signals. Consequently, a treatment for the chemical deglycation with trifluoromethanesulfonic acid was applied, which resulted in the identification of protein Z4, LTP1, CMb, CMe, pUP13, 3a, and Bwiph as constituents of the haze proteome. Because only one hordein was detectable and the proline content in haze hydrolysates was lower than those of barley prolamins, our results suggest that this class of proteins is of minor importance for haze development.

Purification and Structural Characterization of Protein Z4 from Malt

In beer quality, the interactions of proteins with hop acids are understood to be the basis of beer foam stability. Among beer proteins, one barley malt albumin, protein Z4, has an important role in stabilizing beer foam. In this study, protein Z4 was purified from AC Metcalfe barley malt for further study. The secondary structure of protein Z4 was investigated via Fourier transform infrared spectroscopy and circular dichroism spectroscopy. The results showed that protein Z4 had high structural stability. These structural features of protein Z4 were proposed to explain its observed high tolerance and stability of high temperatures and pH during the brewing process. Relatively high beer ethanol concentrations and yeast-induced modifications during the fermentation process can only partially influence in the structure of protein Z4. Modification by carbohydrates was observed as a result of the malting process. Meanwhile, modifications could increase the stability of the structure of protein Z4. Furthermore, modifications to protein Z4 were also conducive to improving its contribution to beer foam stability. Finally, these results confirmed the positive relationship between protein Z4 and beer foam stability.

Heterologous expression of Hordeum vulgare protein Z4 in Pichia pastoris shows increased structural stability

Protein Z4, an abundant of serpin storage proteins of Hordeum vulgare, is one major factor that benefits the stability of beer foam. In this study, protein Z4 from Canadian malt Metcalfe was cloned and expressed in Pichia pastoris. The glycosylated 45kDa of recombinant protein Z4 was purified by Ni-NTA affinity column. The secondary structure of recombinant protein Z4 was detected via Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR), and β-sheets and β-turns were the main structure of recombinant protein Z4. A few structural differences between recombinant protein Z4 and protein Z4 from malt were also analyzed, that more obvious α-helical structure and more S-H contents were detected in recombinant protein Z4. Meanwhile, higher structural stability of recombinant protein Z4 was also observed after incubating the samples in a wide range of temperature, pH and ethanol. These results confirmed the positive relationship between modifications by saccharides and high structural stability of recombinant protein Z4.

Characterization of barley serpin Z7 that plays multiple roles in malt and beer.

Barley protein Z7 (BSZ7) is a well-known serine protease inhibitor that was regarded as a major effector of beer foam stability. Moreover, it has also been suggested to participate in haze formation and affect wort filterability. The present study purified BSZ7 from barley malt and characterized its secondary structure and modification, as well as its relationship with peroxidase, to elucidate the molecular base of BSZ7 that supports its multiple roles in malt and beer. It was found that after 30 min of heating, the secondary structure was not affected. BSZ7 has no inhibiting effect on nonspecific protease originated from malt, suggesting its negative role in wort filterability was accomplished by other means. Furthermore, the glycation of BSZ7 by the Maillard reaction may make some contribution to its survival during wort boiling. The interaction of BSZ7 with polysaccharides and polyphenols found by adding experiment may explain how it acts as a negative factor on wort filterability. Greater understanding of BSZ7 and other proteins of malts will lead to better improvements in brewing quality.

In vitrostudies on the main beer protein Z4 ofHordeum vulgareconcerning heat stability, protease inhibition and gushing

Protein Z4, the most abundant form of serpin protein Z of Hordeum vulgare, is one of the major constituents of beer foam and inhibits the activity of serine proteases. The possible influence of protein Z4 on malt proteases, which impact malt quality, is of interest for brewers. In addition, the persistence of Z4 in the brewing process and the ensuing presence of Z4 in beer matters to brewers because of its impact on foam. In order to analyse the influence of Z4 on protease inhibition and beer gushing, Pichia pastoris cells were heterologously transformed with the Z4 coding gene and the protein was recovered from the supernatant of a transformant's liquid culture. Agar diffusion assays showed that the recombinant Z4 protein had an inhibitory effect on proteases present in barley malt. Heat denaturation of the protein impaired protease inhibition and revealed degradation of the structure of Z4. The effect of Z4 on hydrophobin-induced gushing was analysed by addition of the protein to beers pre-treated with the class 2 hydrophobin FcHyd5p. Results demonstrated that addition of protein Z4 to gushing beer reduces the overflow volume considerably. Heat treatment again had a negative impact on the gushing reduction capacity of Z4. Copyright © 2014 The Institute of Brewing & Distilling

Mutation Analysis of Barley Malt Protein Z4 and Protein Z7 on Beer Foam Stability

Beer foam stability is an important characteristic. It has been suggested that isoforms of protein Z, that is, protein Z4 and protein Z7, contribute to beer foam stability. We investigated the relationship between beer foam stability and protein Z4 and protein Z7 using their deficient mutants. As a protein Z4-deficient mutant, cv. Pirkka was used. Protein Z7 deficiency was screened in 1564 barley accessions in the world collection of Okayama University, Japan. The barley samples from normal, protein Z4-deficient, protein Z7-deficient, and double-deficient were genotyped in F(2) populations and then pooled based on the DNA marker genotypes of protein Z4 and protein Z7. For a brewing trial, F(5) pooled subpopulations were used. After malting and brewing, the foam stability was determined, and the results showed that the levels of foam stability in the four samples were comparable. Two-dimensional gel electrophoresis was used to investigate the proteome in these beer samples. The results showed that low molecular weight proteins, including lipid transfer protein (LTP2), in the deficient mutants were higher than those in the normal sample. Our results suggest that the contribution of protein Z4 and protein Z7 to beer foam stability was not greater than that of other beer proteins.

The Chriz–Z4 complex recruits JIL-1 to polytene chromosomes,a requirement for interband-specific phosphorylation of H3S10

The conserved band-interband pattern is thought to reflect the looped-domain organization of insect polytene chromosomes. Previously, we have shown that the chromodomain protein Chriz and the zinc-finger protein Z4 are essentially required for the maintenance of polytene chromosome structure. Here we show that both proteins form a complex that recruits the JIL-1 kinase to polytene chromosomes, enabling local H3S10 phosphorylation of interband nucleosomal histones. Interband targeting domains were identified at the N-terminal regions of Chriz and Z4, and our data suggest partial cooperation of the complex with the BEAF boundary element protein in polytene and diploid cells. Reducing the core component Chriz by RNAi results in destabilization of the complex and a strong reduction of interband-specific histone H3S10 phosphorylation.

The Effects of Hop-α-Acids and Proline-Specific Endoprotease (PSEP) Treatments on the Foam Quality of Beer

Influences on foam stability and cling were compared by brewing trials investigating beer hopping rate, hopping type and modification of beer protein composition by the inclusion of a proline specific protease (PSEP). The comparison of the NIBEM, Rudin and lacing foam assessment methods with the level of hopping demonstrated the superiority of hydrogenated hop alpha-acids with respect to foam stability and particularly lacing. In addition, the NIBEM and Rudin foam analysis tests appear to respond relatively similarly with respect to hopping rate and hop type, with the NIBEM being somewhat more responsive in terms of foam stability measurments. The PSEP trials suggested that protein composition may only have a subtle effect on foam stability. Although more specific to haze active proteins, PSEP treatment in the small and pilot scale trials generally, but not always, resulted in a minor reduction in foam stability. This effect was not observed in 20 hL pilot and industrial scale beer productions. It was verified that both NIBEM and Rudin were positively influenced by increased levels of foam positive proteins. Although both foam tests were responsive to hopping rate and type, it is suggested that the Rudin foam test is somewhat biased towards foam positive proteins, particularly albuminous foam positive proteins (LTP1 and protein Z4), while in comparison the NIBEM foam test appears somewhat biased towards hordein foam positive proteins.

Development of DNA markers associated with beer foam stability for barley breeding

Traits conferring brewing quality are important objectives in malting barley breeding. Beer foam stability is one of the more difficult traits to evaluate due to the requirement for a relatively large amount of grain to be malted and then the experimental costs for subsequent brewing trials. Consequently, foam stability tends to be evaluated with only advanced lines in the final stages of the breeding process. To simplify the evaluation and selection for this trait, efficient DNA makers were developed in this study. Previous studies have suggested that the level of both of the foam-associated proteins Z4 and Z7 were possible factors that influenced beer foam stability. To confirm the relationship between levels of these proteins in beer and foam stability, 24 beer samples prepared from malt made from 10 barley cultivars, were examined. Regression analyses suggested that beer proteins Z4 and Z7 could be positive and negative markers for beer foam stability, respectively. To develop DNA markers associated with contents of proteins Z4 and Z7 in barley grain, nucleotide sequence polymorphisms in barley cultivars in the upstream region of the translation initiation codon, where the promoter region might be located were compared. As a result, 5 and 23 nucleotide sequence polymorphisms were detected in protein Z4 and protein Z7, respectively. By using these polymorphisms, cleaved amplified polymorphic sequence (CAPS) markers were developed. The CAPS markers for proteins Z4 and Z7 were applied to classify the barley grain content of 23 barley cultivars into two protein Z4 (pZ4-H and pZ4-L) and three protein Z7 (the pZ7-H, pZ7-L and pZ7-L2) haplotypes, respectively. Barley cultivars with pZ4-H showed significantly higher levels of protein Z4 in grain, and those with pZ7-L and pZ7-L2 showed significantly lower levels of protein Z7 in grain. Beer foam stability in the cultivars with pZ4-H and pZ7-L was significantly higher than that with pZ4-L and pZ7-H, respectively. Our results indicate that these CAPS markers provide an efficient selection tool for beer foam stability in barley breeding programs.

Identification of novel haze-active beer proteins by proteome analysis

Colloidal haze reduces beer quality considerably. Four haze samples were analyzed by two-dimensional gel electrophoresis (2DE) in order to identify haze-active proteins. Several protein spots were observed in all of the four haze samples. Using mass spectrometry analysis followed by a database search identified these spots as barley dimeric alpha-amylase inhibitor (BDAI-1), CMb component of tetrameric alpha-amylase inhibitor (CMb) and trypsin inhibitor CMe precursor (CMe). These proteins were considered to be haze-active. Since haze-active proteins are adsorbed by silica gel in the beer filtration process, we eluted proteins adsorbed onto silica gel (PAS) and identified their species. These major PAS were identified as protein Z4, protein Z7 and trypsin/amylase inhibitor pUP13 (TAI), rather than BDAI-1, CMb and CMe. Furthermore, we analyzed proline compositions in the beer proteins, PAS and the haze proteins. Consequently, we found that the proline compositions of PAS were higher (ca. 20 mol%) than those in the beer proteins (ca. 10 mol%), although those of the haze-active proteins such as BDAI-1, CMb and CMe were 6.6–8.7 mol%. Our results suggest that BDAI-1, CMb and CMe are not predominant haze-active proteins, but growth factors of beer colloidal haze.

Comparison of Foam Quality and the Influence of Hop α-Acids and Proteins Using Five Foam Analysis Methods

Rudin, NIBEM, lacing index, cylinder pour, and shake foam quality tests were comprehensively compared to evaluate their utility and responsiveness to beer components that are thought to influence foam quality. Each of the tests responded differently to beer characteristics that influence beer foam quality. The Rudin and shake tests responded positively to the level and content of foam-promoting proteins (i.e., protein Z4) and also to the level of isomerized hop α-acids in beer. However, the shake test did not differentiate between isomerized and hydrogenated hop α-acids, whereas the Rudin test did. The lacing index responded positively to isomerized hop α-acids, hydrogenated in particular, but possibly negatively to alcohol. Hydrogenation of hop α-acids and protein Z4 increased foam stability as measured by the cylinder pour test, whereas alcohol, higher pH, and CO2 level tended to reduce foam stability. A positive association with the level of isomerized hop acids was not observed; however, further investigation is required to clarify this. Finally, the NIBEM test results were influenced positively by the level of isomerized hop acids and protein Z4, while they were negatively correlated with viscosity and final gravity. Hop α-acid hydrogenation substantially improved NIBEM foam stability compared with isomerized hop α-acids. The NIBEM test was correlated positively with the Rudin, cylinder pour, lacing index, and shake (foam quality generated) tests. These comparisons and observations suggest how these foam quality evaluation tests may be best used for quality control/assurance and beverage development.

MSL Complex Is Attracted to Genes Marked by H3K36 Trimethylation Using a Sequence-Independent Mechanism

In Drosophila, X chromosome dosage compensation requires the male-specific lethal (MSL) complex, which associates with actively transcribed genes on the single male X chromosome to upregulate transcription approximately 2-fold. We found that on the male X chromosome, or when MSL complex is ectopically localized to an autosome, histone H3K36 trimethylation (H3K36me3) is a strong predictor of MSL binding. We isolated mutants lacking Set2, the H3K36me3 methyltransferase, and found that Set2 is an essential gene in both sexes of Drosophila. In set2 mutant males, MSL complex maintains X specificity but exhibits reduced binding to target genes. Furthermore, recombinant MSL3 protein preferentially binds nucleosomes marked by H3K36me3 in vitro. Our results support a model in which MSL complex uses high-affinity sites to initially recognize the X chromosome and then associates with many of its targets through sequence-independent features of transcribed genes.

Identification of the Major Proteins in Beer Foam by Mass Spectrometry following Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Previously unknown beer and beer foam proteins of barley origin were identified by tandem mass spectrometry (MS) sequencing after in-gel tryptic digestion. Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis 40-kDa and 7- to 17-kDa bands were excised for these analyses. In total, there were 30 proteins detected, 21 proteins found in beer, and 24 proteins found in beer foam including the common 15 proteins in beer and 2× foam. Most originated from the barley albumins and were resistant to enzymes during brewing. There were also two hordein-related proteins. The proteins identified enlarge and update the list of proteins related to foam, usually confined to protein Z4, lipid transfer protein 1 (LTP1), and some hordein-derived proteins.

Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster polytene chromosomes

Polytene interphase chromosomes are compacted into a series of bands and interbands reflecting their organization into independent chromosomal domains. In order to understand chromosomal organization, we set out to study the role of proteins that are selective for interbands. Here we describe the Drosophila melanogaster chromodomain protein Chriz that is coimmunoprecipitated with the zinc finger protein Z4. Both proteins colocalize exclusively to the interbands on Drosophila polytene chromosomes. Like Z4, Chriz is ubiquitously expressed throughout development and is associated with chromatin in all interphase nuclei. Following dissociation from chromatin, early in mitosis Chriz binds to the centrosomes and to the mitotic spindle. Newly induced amorphic Chriz alleles are early lethal, and ubiquitous overexpression of Chriz is lethal as well. Available Chriz hypomorphs which survive until pupal stage have a normal chromosomal phenotype. Reducing Z4 protein does not affect Chriz binding to polytene chromosomes and vice versa. Z4 is still chromosomally bound when Chriz protein is depleted by RNA interference.

Identification of the Drosophila interband-specific protein Z4 as a DNA-binding zinc-finger protein determining chromosomal structure.

The subdivision of polytene chromosomes into bands and interbands suggests a structural chromatin organization that is related to the formation of functional domains of gene expression. We made use of the antibody Z4 to gain insight into this level of chromosomal structure, as the Z4 antibody mirrors this patterning by binding to an antigen that is present in most interbands. The Z4 gene encodes a protein with seven zinc fingers, it is essential for fly development and acts in a dose-dependent manner on the development of several tissues. Z4 mutants have a dose-sensitive effect on w(m4) position effect variegation with a haplo-suppressor and triplo-enhancer phenotype, suggesting Z4 to be involved in chromatin compaction. This assumption is further supported by the phenotype of Z4 mutant chromosomes, which show a loss of the band/interband pattern and are subject to an overall decompaction of chromosomal material. By co-immunoprecipitations we identified a novel chromo domain protein, which we named Chriz (Chromo domain protein interacting with Z4) as an interaction partner of Z4. Chriz localizes to interbands in a pattern that is identical to the Z4 pattern. These findings together with the result that Z4 binds directly to DNA in vitro strongly suggest that Z4 in conjunction with Chriz is intimately involved in the higher-order structuring of chromosomes.