Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.     

Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1

There are 10 gene families that have members on both human chromosome 6 (6p21.3, the location of the human major histocompatibility complex [MHC]) and human chromosome 9 (mostly 9q33-34). Six of these families also have members on mouse chromosome 17 (the mouse MHC chromosome) and mouse chromosome 2. In addition, four of these families have members on human chromosome 1 (1q21-25 and 1p13), and two of these have members on mouse chromosome 1. One hypothesis to explain these patterns is that members of the 10 gene families of human chromosomes 6 and 9 were duplicated simultaneously as a result of polyploidization or duplication of a chromosome segment ("block duplication"). A subsequent block duplication has been proposed to account for the presence of representatives of four of these families on human chromosome 1. Phylogenetic analyses of the 9 gene families for which data were available decisively rejected the hypothesis of block duplication as an overall explanation of these patterns. Three to five of the genes on human chromosomes 6 and 9 probably duplicated simultaneously early in vertebrate history, prior to the divergence of jawed and jawless vertebrates, and shortly after that, all four of the genes on chromosomes 1 and 9 probably duplicated as a block. However, the other genes duplicated at different times scattered over at least 1.6 billion years. Since the occurrence of these clusters of related genes cannot be explained by block duplication, one alternative explanation is that they cluster together because of shared functional characteristics relating to expression patterns.      

Evolutionary distances for protein-coding sequences: modeling site- specific residue frequencies

Estimation of evolutionary distances from coding sequences must take into account protein-level selection to avoid relative underestimation of longer evolutionary distances. Current modeling of selection via site-to-site rate heterogeneity generally neglects another aspect of selection, namely position-specific amino acid frequencies. These frequencies determine the maximum dissimilarity expected for highly diverged but functionally and structurally conserved sequences, and hence are crucial for estimating long distances. We introduce a codon- level model of coding sequence evolution in which position-specific amino acid frequencies are free parameters. In our implementation, these are estimated from an alignment using methods described previously. We use simulations to demonstrate the importance and feasibility of modeling such behavior; our model produces linear distance estimates over a wide range of distances, while several alternative models underestimate long distances relative to short distances. Site-to-site differences in rates, as well as synonymous/nonsynonymous and first/second/third-codon-position differences, arise as a natural consequence of the site-to-site differences in amino acid frequencies.      

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

A Ribonucleoprotein Complex Protects the Interleukin-6 mRNA from Degradation by Distinct Herpesviral Endonucleases

During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3’ untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3’ UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3’ UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.     

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest‐4‐en‐3‐one

The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high‐resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side‐chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl‐branched phthiocerol dimycocerosates. We observed that cholesterol‐exposed cells lacking CYP125A1 accumulate cholest‐4‐en‐3‐one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest‐4‐en‐3‐one and cholesterol, efficiently hydroxylates both of them at C‐27, and then further oxidizes 27‐hydroxycholest‐4‐en‐3‐one to cholest‐4‐en‐3‐one‐27‐oic acid. We determined the X‐ray structure of cholest‐4‐en‐3‐one‐bound CYP125A1 at a resolution of 1.58 Å. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest‐4‐en‐3‐one. In its absence, the latter compound is toxic for both CDC1551 and H37Rv when added with glycerol as a second carbon source. CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest‐4‐en‐3‐one.     

The role of lateral roots in bypass flow in rice (Oryza sativa L.)

Although an apoplastic pathway (the so‐called bypass flow) is implicated in the uptake of Na⁺ by rice growing in saline conditions, the point of entry of this flow into roots remains to be elucidated. We investigated the role of lateral roots in bypass flow using the tracer trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) and the rice cv. IR36. PTS was identified in the vascular tissue of lateral roots using both epifluorescence microscopy and confocal laser scanning microscopy. Cryo‐scanning electron microscopy and epifluorescence microscopy of sections stained with berberine‐aniline blue revealed that the exodermis is absent in the lateral roots. We conclude that PTS can move freely through the cortical layers of lateral roots, enter the stele and be transported to the shoot via the transpiration stream.