Characterization of human β,β-carotene-15,15′-monooxygenase (BCMO1) as a soluble monomeric enzyme

The formal first step in in vitamin A metabolism is the conversion of its natural precursor β,β-carotene (C40) to retinaldehyde (C20). This reaction is catalyzed by the enzyme β,β-carotene-15,15′-monooxygenase (BCMO1). BCMO1 has been cloned from several vertebrate species, including humans. However, knowledge about this protein’s enzymatic and structural properties is scant. Here we expressed human BCMO1 in Spodoptera frugiperda 9 insect cells. Recombinant BCMO1 is a soluble protein that displayed Michaelis–Menten kinetics with a K_M of 14 μM for β,β-carotene. Though addition of detergents failed to increase BCMO1 enzymatic activity, short chain aliphatic detergents such as C₈E₄ and C₈E₆ decreased enzymatic activity probably by interacting with the substrate binding site. Thus we purified BCMO1 in the absence of detergent. Purified BCMO1 was a monomeric enzymatically active soluble protein that did not require cofactors and displayed a turnover rate of about 8 molecules of β,β-carotene per second. The aqueous solubility of BCMO1 was confirmed in mouse liver and mammalian cells. Establishment of a protocol that yields highly active homogenous BCMO1 is an important step towards clarifying the lipophilic substrate interaction, reaction mechanism and structure of this vitamin A forming enzyme.     

Senescence marker protein 30 inhibits angiotensin II-induced cardiac hypertrophy and diastolic dysfunction

Background and objective

Senescence marker protein 30 (SMP30) is assumed to behave as an anti-aging factor. Recently, we have demonstrated that deficiency of SMP30 exacerbates angiotensin II-induced cardiac hypertrophy, dysfunction and remodeling, suggesting that SMP30 may have a protective role in the heart. Thus, this study aimed to test the hypothesis that up-regulation of SMP30 inhibits cardiac adverse remodeling in response to angiotensin II.

Methods

We generated transgenic mice with cardiac-specific overexpression of SMP30 gene using α-myosin heavy chain promoter. Transgenic mice and wild-type littermate mice were subjected to continuous angiotensin II infusion (800 ng/kg/min).

Results

After 14 days, heart weight and left ventricular weight were lower in transgenic mice than in wild-type mice, although blood pressure was similarly elevated during angiotensin II infusion. Cardiac hypertrophy and diastolic dysfunction in response to angiotensin II were prevented in transgenic mice compared with wild-type mice. The degree of cardiac fibrosis by angiotensin II was lower in transgenic mice than in wild-type mice. Angiotensin II-induced generation of superoxide and subsequent cellular senescence were attenuated in transgenic mouse hearts compared with wild-type mice.

Conclusions

Cardiac-specific overexpression of SMP30 inhibited angiotensin II-induced cardiac adverse remodeling. SMP30 has a cardio-protective role with anti-oxidative and anti-aging effects and could be a novel therapeutic target to prevent cardiac hypertrophy and remodeling due to hypertension.     

Ubiquitin E3 ligase SCF regulates TRIB2 stability in liver cancer cells

Tribbles homolog 2 (TRIB2) is functionally important for liver cancer cell survival and transformation. Our previous study demonstrates TRIB2 is stable in liver cancer cells due to the impaired ubiquitination by Smurf1. However, overexpression of Smurf1 alone cannot completely abolish TRIB2 protein expression, whether other potential factors involved in the degradation of TRIB2 still remains unclear. In the present study, we reveal that the stability and ubiquitination of TRIB2 can also be controlled by ubiquitin E3 ligase SCF^β-TRCP. Depletion of either Cullin1 or β-TRCP up-regulates TRIB2 protein expression. Moreover, knockdown of β-TRCP extends the half-life, whereas reduces ubiquitylation of TRIB2. Similar to Smurf1, β-TRCP exerts its role through the TRIB2 Degradation Domain (TDD) at the N-terminus of the TRIB2 protein. Hence, we add TRIB2 to the substrate list of SCF^β-TRCP and the finding may be helpful in the treatment of TRIB2 dependent liver cancer.     

Two β-strands of RAGE participate in the recognition and transport of amyloid-β peptide across the blood brain barrier

Amyloid-β (Aβ) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aβ peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two β-strands of RAGE participate in the interaction with Aβ peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth β-strands are required for interaction with Aβ peptide. Site-directed mutagenesis of amino acids located in the third and eighth β-strands abolish the interaction of RAGE with Aβ peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aβ peptide treatment, while a RAGE mutant defective in Aβ binding does not. Furthermore, use of peptide for the third β-strand or a RAGE monoclonal antibody that targets the RAGE–Aβ interaction interface inhibited transport of the Aβ peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.     

Stain-Free total protein staining is a superior loading control to β-actin for Western blots

Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots.     

Aβ Monomers Transiently Sample Oligomer and Fibril-Like Configurations: Ensemble Characterization Using a Combined MD/NMR Approach

Amyloid β (Aβ) peptides are a primary component of fibrils and oligomers implicated in the etiology of Alzheimer's disease (AD). However, the intrinsic flexibility of these peptides has frustrated efforts to investigate the secondary and tertiary structure of Aβ monomers, whose conformational landscapes directly contribute to the kinetics and thermodynamics of Aβ aggregation. In this work, de novo replica exchange molecular dynamics (REMD) simulations on the microseconds-per-replica timescale are used to characterize the structural ensembles of Aβ42, Aβ40, and M35-oxidized Aβ42, three physiologically relevant isoforms with substantially different aggregation properties. J-coupling data calculated from the REMD trajectories were compared to corresponding NMR-derived values acquired through two different pulse sequences, revealing that all simulations converge on the order of hundreds of nanoseconds-per-replica toward ensembles that yield good agreement with experiment. Though all three Aβ species adopt highly heterogeneous ensembles, these are considerably more structured compared to simulations on shorter timescales. Prominent in the C-terminus are antiparallel β-hairpins between L17–A21, A30–L36, and V39–I41, similar to oligomer and fibril intrapeptide models that expose these hydrophobic side chains to solvent and may serve as hotspots for self-association. Compared to reduced Aβ42, the absence of a second β-hairpin in Aβ40 and the sampling of alternate β topologies by M35-oxidized Aβ42 may explain the reduced aggregation rates of these forms. A persistent V24–K28 bend motif, observed in all three species, is stabilized by buried backbone to side-chain hydrogen bonds with D23 and a cross-region salt bridge between E22 and K28, highlighting the role of the familial AD-linked E22 and D23 residues in Aβ monomer folding. These characterizations help illustrate the conformational landscapes of Aβ monomers at atomic resolution and provide insight into the early stages of Aβ aggregation pathways.     

Conservation and Variability in the Structure and Function of the Cas5d Endoribonuclease in the CRISPR-Mediated Microbial Immune System

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR–Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event.     

What does make an amyloid toxic: Morphology,structure or interaction with membrane?

The toxicity of amyloids is a subject under intense scrutiny. Many studies link this toxicity to the existence of various intermediate structures prior to the fiber formation and/or their specific interaction with membranes. Membranes can also be a catalyst of amyloidogenesis and the composition or the charge of membrane lipids may be of particular importance. Despite intensive research in the field, such intermediates are not yet fully characterized probably because of the lack of adapted methods for their analyses, and the mechanisms of interaction with the membrane are far to be understood. The purpose of this mini-review is to highlight some in vitro characteristics that seem to be convergent to explain the toxicity observed for some amyloids. Based on a comparison between the behavior of a model non-toxic amyloid (the Prion Forming Domain of HET-s) and its toxic mutant (M8), we could establish that short oligomers and/or fibers assembled in antiparallel β-sheets strongly interact with membrane leading to its disruption. Many recent evidences are in favor of the formation of antiparallel toxic oligomers assembled in β-helices able to form pores. We may also propose a new model of amyloid interaction with membranes by a “raft-like” mode of insertion that could explain important destabilization of membranes and thus amyloid toxicity.