Cellular prion protein regulates its own α-cleavage through ADAM8 in skeletal muscle

The ubiquitously expressed cellular prion protein (PrP(C)) is subjected to the physiological α-cleavage at a region critical for both PrP toxicity and the conversion of PrP(C) to its pathogenic prion form (PrP(Sc)), generating the C1 and N1 fragments. The C1 fragment can activate caspase 3 while the N1 fragment is neuroprotective. Recent articles indicate that ADAM10, ADAM17, and ADAM9 may not play a prominent role in the α-cleavage of PrP(C) as previously thought, raising questions on the identity of the responsible protease(s). Here we show that, ADAM8 can directly cleave PrP to generate C1 in vitro and PrP C1/full-length ratio is greatly decreased in the skeletal muscles of ADAM8 knock-out mice; in addition, the PrP C1/full-length ratio is linearly correlated with ADAM8 protein level in myoblast cell line C2C12 and in skeletal muscle tissues of transgenic mice. These results indicate that ADAM8 is the primary protease responsible for the α-cleavage of PrP(C) in muscle cells. Moreover, we found that overexpression of PrP(C) led to up-regulation of ADAM8, suggesting that PrP(C) may regulate its own α-cleavage through modulating ADAM8 activity.     

Toward convergence of experimental studies and theoretical modeling of the chromatin fiber

Understanding the structural organization of eukaryotic chromatin and its control of gene expression represents one of the most fundamental and open challenges in modern biology. Recent experimental advances have revealed important characteristics of chromatin in response to changes in external conditions and histone composition, such as the conformational complexity of linker DNA and histone tail domains upon compact folding of the fiber. In addition, modeling studies based on high-resolution nucleosome models have helped explain the conformational features of chromatin structural elements and their interactions in terms of chromatin fiber models. This minireview discusses recent progress and evidence supporting structural heterogeneity in chromatin fibers, reconciling apparently contradictory fiber models.     

The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the α1, α2, and α3 chains of NC2, a stable heterotrimer with a disulfide bridge between α1 and α3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.     

Alkaline Ceramidase 2 (ACER2) and Its Product Dihydrosphingosine Mediate the Cytotoxicity of N-(4-Hydroxyphenyl)retinamide in Tumor Cells

Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.     

Analysis of Ca2+/Mg2+ selectivity in α‐lactalbumin and Ca2+‐binding lysozyme reveals a distinct Mg2+‐specific site in lysozyme

The triggering of Ca²⁺ signaling pathways relies on Ca²⁺/Mg²⁺ specificity of proteins mediating these pathways. Two homologous milk Ca²⁺‐binding proteins, bovine α‐lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest “four‐state” scheme of metal‐ and temperature‐induced structural changes in a protein. The association of Ca²⁺/Mg²⁺ by native proteins is entropy‐driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca²⁺ and Mg²⁺, due to low thermal stabilities of their apo‐forms and relatively high unfavorable enthalpies of Mg²⁺ association. The ratios of their apparent affinities to Ca²⁺ and Mg²⁺, being unusually high at low temperatures (5.3–6.5 orders of magnitude), reach the values inherent to classical EF‐hand motifs at physiological temperatures. The comparison of phase diagrams predicted within the model of competitive Ca²⁺ and Mg²⁺ binding with experimental data strongly suggests that the association of Ca²⁺ and Mg²⁺ ions with bLA is a competitive process, whereas the primary Mg²⁺ site of EQL is different from its Ca²⁺‐binding site. The later conclusion is corroborated by qualitatively different molar ellipticity changes in near‐UV region accompanying Mg²⁺ and Ca²⁺ association. The Ca²⁺/Mg²⁺ selectivity of Mg²⁺‐site of EQL is below an order of magnitude. EQL exhibits a distinct Mg²⁺‐specific site, probably arising as an adaptation to the extracellular environment. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structural refinement of the hERG1 pore and voltage‐sensing domains with ROSETTA‐membrane and molecular dynamics simulations

The hERG1 gene (Kv11.1) encodes a voltage‐gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template‐driven de‐novo design with ROSETTA‐membrane modeling using side‐chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2‐Kv2.1 chimera channels, the missing parts are modeled de‐novo. The impact of several alignments on the structure of the S4 helix in the voltage‐sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage‐sensor domain; and the topology of the extracellular S5‐pore linker compared with that established by toxin foot‐printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.