Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study

<p>Abstract</p> <p>Background</p> <p>Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR) to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity.</p> <p>Results</p> <p>We show that DNA -bound salmon (salmine) and squid protamines contain <it>α</it>-helix, <it>β</it>-turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No <it>β</it>-sheet was observed. In salmine, the <it>α</it>-helix amounted to ~20%, while in squid protamine it reached ~40%. In contrast, the structure not stabilized by intermolecular hydrogen bonding was more abundant in salmine (~40%) than in squid protamine (~20%). Both protamines contained ~40% <it>β</it>-turns. The different helical potential of salmine and squid protamine was confirmed by structure predictions and CD in the presence of trifluoroethanol.</p> <p>Conclusion</p> <p>DNA-bound protamine in sperm nuclei contains large amounts of defined secondary structure stabilized by intramolecular hydrogen bonding. Both salmine and squid protamine contain similar amounts of <it>β</it>-turns, but differ in the proportions of <it>α</it>-helix and non-hydrogen bonded conformations. In spite of the large differences in the proportions of secondary structure motifs between salmon and squid protamines, they appear to be equally efficient in promoting tight hexagonal packing of the DNA molecules in sperm nuclei.</p

Phosphorylation of the carboxy-terminal domain of histone H1: effects on secondary structure and DNA condensation

Linker histone H1 plays an important role in chromatin folding. Phosphorylation by cyclin-dependent kinases is the main post-translational modification of histone H1. We studied the effects of phosphorylation on the secondary structure of the DNA-bound H1 carboxy-terminal domain (CTD), which contains most of the phosphorylation sites of the molecule. The effects of phosphorylation on the secondary structure of the DNA-bound CTD were site-specific and depended on the number of phosphate groups. Full phosphorylation significantly increased the proportion of β-structure and decreased that of α-helix. Partial phosphorylation increased the amount of undefined structure and decreased that of α-helix without a significant increase in β-structure. Phosphorylation had a moderate effect on the affinity of the CTD for the DNA, which was proportional to the number of phosphate groups. Partial phosphorylation drastically reduced the aggregation of DNA fragments by the CTD, but full phosphorylation restored to a large extent the aggregation capacity of the unphosphorylated domain. These results support the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of H1 partial phosphorylation in interphase chromatin relaxation. More generally, our results suggest that the effects of phosphorylation are mediated by specific structural changes and are not simply a consequence of the net charge

Histone H1 Post-Translational Modifications : Update and Future Perspectives

Histone H1 is the most variable histone and its role at the epigenetic level is less characterized than that of core histones. In vertebrates, H1 is a multigene family, which can encode up to 11 subtypes. The H1 subtype composition is different among cell types during the cell cycle and differentiation. Mass spectrometry-based proteomics has added a new layer of complexity with the identification of a large number of post-translational modifications (PTMs) in H1. In this review, we summarize histone H1 PTMs from lower eukaryotes to humans, with a particular focus on mammalian PTMs. Special emphasis is made on PTMs, whose molecular function has been described. Post-translational modifications in H1 have been associated with the regulation of chromatin structure during the cell cycle as well as transcriptional activation, DNA damage response, and cellular differentiation. Additionally, PTMs in histone H1 that have been linked to diseases such as cancer, autoimmune disorders, and viral infection are examined. Future perspectives and challenges in the profiling of histone H1 PTMs are also discussed

Characterization of the secondary structure of histone h1 subtypes by circular dichroism (cd)

Introducción: Las histonas H1 modulan la estructura y la función de la cromatina. Las células somáticas de mamífero contienen los subtipos H1°, H1a, H1b, H1c, H1d y H1e; en células germinales de testículo y en ovocito, se encuentran respectivamente H1t y H1oo. Su estructura está conformada por un dominio central globular flanqueado por los dominios N-Terminal (DNT) y C-Terminal (DCT). Objetivo: Caracterizar la estructura secundaria de subtipos de la histona H1 mediante dicroísmo circular (DC). Materiales y Métodos: La histona H1 total se extrajo de núcleos de cerebro de rata por cromatografía de intercambio catiónico; la H1° se purificó por filtración en gel y las H1a, H1b, H1c y H1e por cromatografía líquida de alta resolución de fase reversa (RF-HPLC). Los espectros de DC se realizaron en tampón fosfato 10 mM; tampón fosfato 10 mM, 20% TFE (trifluoroetanol); tampón fosfato 10 mM, 40% TFE; tampón fosfato 10 mM, 60% TFE; tampón fosfato 10 mM, 150 mM NaCl y tampón fosfato 10 mM, 1 M NaCl. El análisis de los espectros se realizó con el programa Standard Analysis. Resultados: El porcentaje de hélice-alfa se calculó por diferentes métodos matemáticos teniendo en cuenta elipticidad molar a 193 nm y a 222 nm; con programa de deconvolución K2D y con relaciones cualitativas R1 y R2. El TFE induce la estructura en hélice-alfa en cada uno de los subtipos, mientras que NaCl no induce ningún cambio importante. Conclusión: Los subtipos con mayor contenido de hélice-alfa son H1a y H1c. Las diferencias observadas en el porcentaje de hélice-alfa entre los diferentes subtipos puede ser importante para su diferenciación funcional.H1 histones modulate the structure and function of chromatin. Mammalian somatic cells contain H1°, H1a, H1b, H1c, H1d and H1e subtypes; H1t and H1oo are found in testicular germ cells and oocyte, respectively. Its structure consists of a globular core domain flanked by N-terminal (DNT) and C-terminal (DCT) domains. Objective: To characterize the secondary structure of histone H1 subtypes through circular dichroism (CD). Materials and Methods: Total histone H1 was extracted for rat brain nuclei by cation exchange chromatography; histone H1° was purified by gel filtration and the histones H1a, H1b, H1c and H1e were purified by reversed phase high performance liquid chromatography (RP-HPLC). CD spectra were performed in 10 mM phosphate buffer; 10 mM, 20% TFE phosphate buffer (trifluoroethanol); 10 mM, 40% TFE; phosphate buffer 10 mM, 60% TFE; phosphate buffer 10 mM, 150 mM NaCl and phosphate buffer 10 mm, 1 M NaCl. The analysis of the spectra was performed with JASCO Standard Analysis. Results: The percentage of alpha-helix was calculated using different mathematical methods, taking into account the molar ellipticity at 193 nm, and 222 nm, with K2D deconvolution program and the R1 and R2 qualitative relationships. The results indicate that TFE induced the alpha-helix structure in each of the subtypes, whereas NaCl did not induce any significant change. Conclusion: H1a and H1c are subtypes with highest content of alpha-helix. The observed differences in the percentage of alpha-helix between different subtypes may be important for their functional differentiation

Smallholder Farmers in the Speciality Coffee Industry: Opportunities, Constraints and the Businesses that are Making it Possible

Coffee has traditionally been a commodity product sold in a highly competitive and saturated global market. This lack of product differentiation has made coffee farmers very vulnerable to fluctuating prices. During the last decade, the coffee industry is undergoing a process of decommoditisation, offering an opportunity for farmers to differentiate their coffee in terms of sustainability and quality and to commercialise it more directly. However, smallholder farmers face productivity and transactional constraints that inhibit them from accessing these higher?value market segments. Intermediaries are needed to connect them with this new market. In this article, we present a cross?case study analysis of three ‘connective businesses’ that are facilitating direct trade relationships between smallholder farmers and speciality coffee roasters

Expression, structure and evolution of H1 linker histones

H1 linker histones bind to linker DNA and contribute to the stabilization of both the nucleosome and chromatin higherorder structure. We have studied aspects of the expression, structure and evolution of H1 linker histones. H1 presents multiple isoforms. In rat cerebral cortex neurons, the subtypes H1a,b,c,d are replaced, to varying degrees, by H1e during postnatal development. In vivo labelling experiments have shown the importance of differential turnover and synthesis rates in defining the H1a-e subtype composition of chromatin. H1o is regulated in a different way, its expression being linked to neuronal terminal differentiation; it is also the only histone subtype which is regulated by external signals in specific neuronal populations. We estimated the rates of nucleotide substitution for several H1 subtypes. The rates of nonsynonymous substitution differed among subtypes by almost one order of magnitude. Such a wide variation in the tolerance of amino acid substitutions is consistent with the functional differentiation of the subtypes. We showed, using CD, 1H-NMR and IR spectroscopy, that the H1 terminal domains acquire substantial amounts of secondary structure upon interaction with DNA. In H1o, we characterized an ahelical element in the N-terminal domain and a helix-turn motif in the C-terminal domain, both adjacent to the globular domain. The N-terminal domain of H1e contains two á-helices separated by a Gly-Gly motif, which behaves as a flexible linker.Hem estudiat aspectes de l'expressio, l'estructura i l'evolucio de les histones de la classe H1, que s'uneixen al DNA internucleosomal i participen en l'estabilitzacio del nucleosoma i de la superestructura de la cromatina. Hem estudiat l'expressio dels subtipus de l'H1 a les neurones de l'escorca cerebral de rata. Durant el desenvolupament postnatal, els subtipus H1a, b, c i d son substituits en diferents proporcions per l'H1e, que es converteix en el subtipus majoritari de les neurones madures. Diversos estudis de marcatge en viu han mostrat la importancia del recanvi diferencial, juntament amb les tasses de sintesi, per a determinar les proporcions dels subtipus a la cromatina. L'expressio de l'H1o esta associada a la diferenciacio terminal neuronal. Tambe es l'unic subtipus que respon a senyals externs en determinades poblacions neuronals. Hem estimat les velocitats de substitucio nucleotidica no sinonima de diversos subtipus de l'H1. Les velocitats de substitucio no sinonima varien al voltant d'un ordre de magnitud. Aquestes diferencies de tolerancia de les substitucions d'aminoacids estan a favor de la diferenciacio funcional dels subtipus de l'H1. Hem demostrat, mitjancant DC, 1H-NMR i espectroscopia d'IR, que els dominis terminals de les H1 adquireixen estructura secundaria quan interaccionen amb el DNA. Hem caracteritzat un element - helicoidal en el domini N-terminal de l'H1o i un motiu helicecolze en el domini C-terminal, ambdos contigus al domini globular. El domini N-terminal de l'H1e conte dues helices separades per un motiu Gly-Gly, que fa de frontissa

Sequence conservation of linker histones between chicken and mammalian species

The percent identity matrices of two sequence multiple alignments between linker histones from chicken and mammalian species are described. Linker histone protein sequences for chicken, mouse, rat and humans, available on public databases were used. This information is related to the research article entitled “Identification of novel post-translational modifications in linker histones from chicken erythrocytes”published in the Journal of Proteomics [1]

Histone H1 Favors Folding and Parallel Fibrillar Aggregation of the 1–42 Amyloid‑β Peptide

Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases of the central nervous system. The aggregation of the amyloid-β peptide, Aβ(1–42), is believed to play an important role in the pathogenesis of AD. Histone H1 is found in the cytoplasm of neurons in AD, and it has been shown to interact with aggregated amyloid-β peptides and with amyloid fibrils. We have used Thioflavin T (ThT) fluorescence enhancement, circular dichroism spectroscopy (CD), coprecipitation, and transmission electron microscopy (TEM) to study the interaction of histone H1 with Aβ(1–42). Both freshly prepared (monomeric) Aβ(1–42) and histone H1 solutions showed negative CD bands typical of the random coil. Mixing Aβ(1–42) and histone H1 led to the loss of the random coil, which was replaced mostly by β-structure. Therefore, both Aβ(1–42) and histone H1 behave as intrinsically disordered proteins with coupled binding and folding. Mutual structure induction demonstrates the interaction of Aβ(1–42) and histone H1. The interaction was confirmed by coprecipitation followed by SDS-PAGE. Mutual structure induction was also observed with the H1 terminal domains. Incubation of Aβ(1–42) for 1 week in the presence of histone H1 led to the formation of laminar aggregates and thick bundles, characterized by the parallel association of large numbers of fibrils. The aggregates were particularly large and ordered with the H1 subtype H1.2. Further aging of the complexes led to tight compaction of fibril bundles and to fiber growth. Stabilization of fibril–fibril interactions appeared to be determined by the C-terminal domain of histone H1. In summary, these observations indicate that histone H1 has at least two effects: it helps the folding of Aβ monomers and stabilizes the parallel association of fibrils

Micrococcal nuclease sequencing of porcine sperm suggests enriched co-location between retained histones and genomic regions related to semen quality and early embryo development

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm’s chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements.These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars