Search

Ribosome profiling is a new emerging technology that uses massively parallel amplification of ribosome-protected fragments and next-generation sequencing to monitor translation in vivo with codon resolution. Studies using this approach provide insightful views on the regulation of translation on a global cell-wide level. In this review, we compare different experimental set-ups and current protocols for sequencing data analysis. Specifically, we review the pitfalls at some experimental steps and highlight the importance of standardized protocol for sample preparation and data processing pipeline, at least for mapping and normalization. [ABSTRACT FROM AUTHOR]

Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergentbased reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins. [ABSTRACT FROM AUTHOR]

ATP binding cassette (ABC) transporters form a superfamily of integral membrane proteins involved in translocation of substrates across the membrane driven by ATP hydrolysis. Despite available crystal structures and extensive biochemical data, many open questions regarding their transport mechanisms remain. Therefore, there is a need to explore spectroscopic techniques such as solid state NMR in order to bridge the gap between structural and mechanistic data. In this study, we investigate the feasibility of using Escherichia coli MsbA as a model ABC transporter for solid state NMR studies. We show that optimised solubilisation and reconstitution procedures enable preparing stable and homogenous protein samples. Depending on the duration of solubilisation, MsbA can be obtained in either an apo- or in a native lipid A bound form. Building onto these optimisations, the first promising MAS-NMR spectra with narrow lines have been recorded. However, further sensitivity improvements are required so that complex NMR experiments can be recorded within a reasonable amount of time. We therefore demonstrate the usability of paramagnetic doping for rapid data acquisition and explore dynamic nuclear polarisation as a method for general signal enhancement. Our results demonstrate that solid state NMR provides an opportunity to address important biological questions related to complex mechanisms of ABC transporters. [ABSTRACT FROM AUTHOR]

Despite the effectiveness of the anti-EGFR chimeric antibody (mAb), cetuximab, in treating nasopharyngeal carcinoma (NPC), its efficacy remains variable and often modest. In this study, a full human dual targeted anti-EGFR/HER3 antibody, CA1182, was generated from phage display library. CA1182 was as effective as cetuximab or trastuzumab in inhabiting phosphorylation of EGFR or HER2, but it exhibited as much more potent than cetuximab or trastuzumab. Moreover, our studies showed that CA1182 was significantly more effective than cetuximab in prolonging the survival of severe combined immune deficient mice bearing human NPC, suggesting that it might be a promising therapeutic agent for NPC. [ABSTRACT FROM AUTHOR]

Ras is a prototypical small G-protein and a central regulator of growth, proliferation and differentiation processes in virtually every nucleated cell. As such, Ras becomes engaged and activated by multiple growth factors, mitogens, cytokines or adhesion receptors. Ras activation comes about by changes in the steady-state equilibrium between the inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states of Ras, resulting in the mostly transient accumulation of Ras-GTP. Three decades of intense Ras research have disclosed various families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) as the two principal regulatory elements of the Ras-GDP/GTP loading status. However, with the possible exception of the GEF Sos, we still have only a rudimentary knowledge of the precise role played by many GEF and GAP members in the signalling network upstream of Ras. As for GAPs, we even lack the fundamental understanding of whether they function as genuine signal transducers in the context of growth factor-elicited Ras activation or rather act as passive modulators of the Ras-GDP/GTP cycle. Here we sift through the large body of Ras literature and review the relevant data for understanding the participation and precise role played by GEFs and GAPs in the process of Ras activation. [ABSTRACT FROM AUTHOR]

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein. [ABSTRACT FROM AUTHOR]

Redox signals have emerged as important regulators of cellular physiology and pathology. The advent of redox imaging in vertebrate systems now provides the opportunity to dynamically visualize redox signaling during development and disease. In this review, we summarize recent advances in the generation of genetically encoded redox indicators (GERIs), introduce new redox imaging strategies, and highlight key publications in the field of vertebrate redox imaging. We also discuss the limitations and future potential of in vivo redox imaging in zebrafish and mice. [ABSTRACT FROM AUTHOR]

Misfolded and incorrectly assembled proteins in the secretory pathway are eliminated by ubiquitylation and proteasomal degradation in a process known as ER-associated degradation (ERAD). Retrotranslocation of diverse substrates including misfolded proteins and viruses occurs through channels in the ER membrane, which are also utilized for host cell penetration by A/B class protein toxins such as cholera toxin, ricin or K28. According to the current view, disulfide-bonded proteins must either be reduced or rearranged to ensure translocation competence and entry into the cytosol from the ER. As the underlying mechanisms are still largely mysterious, we here focus on the redox status and disulfide isomerization of ERAD substrates and the role of oxidoreductases in the essential process of ER-to-cytosol retrotranslocation. [ABSTRACT FROM AUTHOR]

Transcriptional enhancers are short (200-1500 base pairs) DNA segments that are able to dramatically boost transcription from the promoter of a target gene. Originally discovered in simian virus 40 (SV40), a small DNA virus, transcription enhancers were soon also found in immunoglobulin genes and other cellular genes as key determinants of cell-type-specific gene expression. Enhancers can exert their effect over long distances of thousands, even hundreds of thousands of base pairs, either from upstream, downstream, or from within a transcription unit. The number of enhancers in eukaryotic genomes correlates with the complexity of the organism; a typical mammalian gene is likely controlled by several enhancers to fine-tune its expression at different developmental stages, in different cell types and in response to different signaling cues. Here, I provide a personal account of how enhancers were discovered more than 30 years ago, and also address the amazing development of the field since then. [ABSTRACT FROM AUTHOR]

MicroRNAs are small non-coding RNAs that partially bind to the 3' untranslated (3'UTR) regions of target genes in animals and regulate protein production of the target transcripts. MiR-103 has been confirmed to play a critical role in lipid metabolism, however, the target genes and signaling pathway regulated by miR-103 is still unclear. In our experiment, we observed a positive function of miR-103 on the adipogenic differentiation of 3T3-L1 pre-adipocyte. Furthermore, we proved that this function of miR-103 worked through activating AKT/mTOR signal pathway and impairing target gene MEF2D. By inhibiting and over-expressing the MEF2D gene, we found that MEF2D had a negative role in regulating adipocyte key genes, and this function of MEF2D could be impaired by miR-103. In conclusion, we found that miR-103 can promote 3T3-L1 cells differentiation by targeting MEF2D and activating AKT/mTOR signal pathway. These results will shed a light on further study of microRNAs. [ABSTRACT FROM AUTHOR]

Human cysteine cathepsins (family C1, clan CA) have long been regarded as ubiquitous household enzymes, primarily involved in the recycling and degradation of proteins in lysosomes. This opinion has changed considerably during recent decades, however, with the demonstration of their involvement in various physiological processes. A growing body of evidence supports the theory that cathepsins play specific functions in lung homeostasis and pathophysiological events such as asthma, lung fibrosis (including idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease (embracing emphysema and chronic bronchitis), silicosis, bronchopulmonary dysplasia or tumor invasion. The objective of this review is to provide an update on the current knowledge of the role of these enzymes in the lung. Particular attention has been paid to the understanding of the role of these proteases and their natural inhibitors, cystatins (family I25, clan IH), in TGF-β1-driven fibrotic processes with an emphasis on lung fibrosis. [ABSTRACT FROM AUTHOR]

New knowledge about the role of the extracellular matrix (ECM) for the regeneration of tissues opens up new opportunities for the design of innovative biomaterials. In 2009, the German Research Foundation (DFG) established the collaborative research center (CRC) TRR67 "Functional biomaterials for controlling healing processes in bone and skin tissue - from material science to clinical application" (https://research.uni-leipzig.de/trr67/) as a joint effort of Leipzig University and Technische Universität Dresden with partners from Jena and the Technische Universität Berlin. This requires the development of novel functional biomaterials, which improve bone and skin regeneration in an aging, multimorbid population. [Extracted from the article]

Recent advances in biology and medicine most often involve applications, which necessitate bioinformatics analyses and tailored approaches for them. The GCB is the worldwide oldest conference on bioinformatics and reflects timely and cutting-edge science over a wide range of developed bioinformatics methods and applications. This issue "Bioinformatics in theory and application" of I Biological Chemistry i highlights the 36th German Conference on Bioinformatics (GCB) organized in Frankfurt am Main from September 14-17th 2020. [Extracted from the article]

A cancer dogma states that inactivation of oncogene(s) can cause cancer remission, implying that oncogenes are the Achilles' heel of cancers. This current model of cancer has kept oncogenes firmly in focus as therapeutic targets and is in agreement with the fact that in human cancers all cancerous cells, with independence of the cellular heterogeneity existing within the tumour, carry the same oncogenic genetic lesions. However, recent studies of the interactions between an oncogene and its target cell have shown that oncogenes contribute to cancer development via developmental reprogramming of the epigenome within the target cell. These results provide the first evidence that carcinogenesis can be initiated by epigenetic stem cell reprogramming, and uncover a new role for oncogenes in the origin of cancer. Here we analyse these evidences and discuss how this vision offers new avenues for developing novel anti-cancer interventions. [ABSTRACT FROM AUTHOR]

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors. [ABSTRACT FROM AUTHOR]

Microbial infection and tissue injury are well established as the two major drivers of inflammation. However, although it is widely accepted that necrotic cell death can trigger or potentiate inflammation, precisely how this is achieved still remains relatively obscure. Certain molecules, which have been dubbed 'damage-associated molecular patterns' (DAMPs) or alarmins, are thought to promote inflammation upon release from necrotic cells. However, the precise nature and relative potency of DAMPs, compared to conventional pro-inflammatory cytokines or pathogen-associated molecular patterns (PAMPs), remains unclear. How different modes of cell death impact on the immune system also requires further clarification. Apoptosis has long been regarded as a non-inflammatory or even anti-inflammatory mode of cell death, but recent studies suggest that this is not always the case. Necroptosis is a programmed form of necrosis that is engaged under certain conditions when caspase activation is blocked. Necroptosis is also regarded as a highly pro-inflammatory mode of cell death but there has been little explicit examination of this issue. Here we discuss the inflammatory implications of necrosis, necroptosis and apoptosis and some of the unresolved questions concerning how dead cells influence inflammatory responses. [ABSTRACT FROM AUTHOR]

Kallikreins (KLKs) are a group of 15 serine proteases encoded by the KLK locus on chromosome 19. Certain single nucleotide variants (SNVs) within the KLK locus have been linked to human disease. Next-generation sequencing of large human cohorts enables reexamination of genomic variation at the KLK locus. We aimed to identify all KLK-related SNVs and examine their impact on gene regulation and function. To this end, we mined KLK SNVs across Ensembl and Exome Variant Server, with exome-sequencing data from 6503 individuals. PolyPhen- 2-based prediction of damaging SNVs and population frequencies of these SNVs were examined. Damaging SNVs were plotted on protein sequence and structure. We identified 4866 SNVs, the largest number of KLK-related SNVs reported. Fourteen percent of noncoding SNVs overlapped with transcription factor binding sites. We identified 602 missense coding SNVs, among which 148 were predicted to be damaging. Nine missense SNVs were common ( > 1% frequency) and displayed significantly different frequencies between European-American and African-American populations. SNVs predicted to be damaging appeared to alter tertiary structure of KLK1 and KLK6. Similarly, these missense SNVs may affect KLK function, resulting in disease phenotypes. Our study represents a mine of information for those studying KLK-related SNVs and their associations with diseases. [ABSTRACT FROM AUTHOR]

Abstract: Human tissue kallikreins (KLKs) represent the largest contiguous group of protease genes within our genome. All 15 KLK genes co-localize within approximately 260 kb in human chromosome 19q13.3–13.4 (14 640 kb→274 990 kb). They are widely expressed in several tissues and mediate a wide range of critical physiological and pathological processes. Despite the recent developments in KLK research, elucidation of their physiological substrate repertoires remains a largely unfulfilled goal. Phage display, positional scanning and combinatorial peptide library screens have provided some valuable insights into the preferred specificities of these powerful enzymes. More recently, advances in proteomic technologies have enabled more systemic approaches towards identification of KLK substrates in a physiological setting. The advent of degradomic technologies has brought to light several putative physiological substrates and has allowed a deeper appreciation of the in vivo functional roles of KLKs. The aim of this review is to provide an overview of the different techniques that have been utilized towards the elucidation of the substrate specificities of these enzymes and elaborate on their emerging in vivo substrates. [ABSTRACT FROM AUTHOR]

Most types of phospholipase D (PLD) from plants contain a C2 domain and are activated by Ca2+ ions. In this study, other metal ions such as Mg2+, La3+, Ce3+, Tb3+ and Y3+ were examined as effectors of recombinantly produced α-type PLD from white cabbage. All the rare earth ions were able to substitute for Ca2+. The activation curves and displacement experiments reflect a 10- to 50-fold higher affinity of PLD for these ions than for Ca2+; however, the maximum activity attained only 36% of that in the presence of Ca2+. Mg2+ displaced Ca2+ without being able to activate PLD. All ions were bound to the substrate micelles consisting of phosphatidyl- p-nitrophenol, Triton X-100 and SDS (1:8:1, by mole). The affinity of rare earth ions to the micelles was 100-fold higher than that of Ca2+ and Mg2+. A conformational change of the enzyme induced by the low affinity but specific binding of Ca2+ ions is concluded to be essential for maximal PLD activity. As demonstrated by the measurement of Tb3+ fluorescence, the substitution of Ca2+ by rare earth ions provides a new avenue for studying the enigmatic role of Ca2+ ions in the modulation of PLD activity in plants. [ABSTRACT FROM AUTHOR]

As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided. [ABSTRACT FROM AUTHOR]

A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure. [ABSTRACT FROM AUTHOR]

The energetics of macromolecular interactions are complex, particularly where protein flexibility is involved. Exploiting serendipitous differences in the plasticity of a series of closely related trypsin variants, we analyzed the enthalpic and entropic contributions accompanying interaction with L45K-eglin C. Binding of the four variants show significant differences in released heat, although the affinities vary little, in accordance with the principle of enthalpy-entropy compensation. Binding of the most disordered variant is almost entirely enthalpically driven, with practically no entropy change. As structures of the complexes reveal negligible differences in protein-inhibitor contacts, we conclude that solvent effects contribute significantly to binding affinities. [ABSTRACT FROM AUTHOR]

Existent nucleic acid isothermal detection techniques for clinical diseases are difficult to promote greatly due to limitations in such aspects as methodology, costs of detection, amplification efficiency and conditions for operation. There is therefore an urgent need for a new isothermal amplification method with the characteristics of high accuracy, easy operation, short time of detection and low costs. We have devised a new method of nucleic acid isothermal amplification using Bst DNA polymerase under isothermal conditions (60-65°C). We call this method of amplification by shortening the distance between forward and reverse primers for nucleic acid isothermal amplification SDAMP. The results demonstrated that this technique is highly sensitive, specific and has short reaction times (40-60 min). Results of sequencing show that the products of SDAMP amplification are mainly polymers formed by series connection of monomers formed through linkage of forward primer and complementary sequences in reverse primer via a few bases. The method is different from current methods of nucleic acid amplification. Our study shows, however, that it is a specific method of nucleic acid isothermal amplification depending on interactions between primers and DNA template. [ABSTRACT FROM AUTHOR]

Human dyskerin is an evolutively conserved protein that participates in diverse nuclear complexes: the H/ACA snoRNPs, that control ribosome biogenesis, RNA pseudouridylation, and stability of H/ACA snoRNAs; the scaRNPs, that control pseudouridylation of snRNAs; and the telomerase active holoenzyme, which safeguards telomere integrity. The biological importance of dyskerin is further outlined by the fact that its deficiency causes the X-linked dyskeratosis congenita disease, while its over-expression characterizes several types of cancers and has been proposed as prognostic marker. The role of dyskerin in telomere maintenance has widely been discussed, while its functions as H/ACA sno/scaRNP component has been so far mostly overlooked and represent the main goal of this review. Here we summarize how increasing evidence indicates that the snoRNA/microRNA pathways can be interlaced, and that dyskerin-dependent RNA pseudouridylation represents a flexible mechanism able to modulate RNA function in different ways, including modulation of splicing, change of mRNA coding properties, and selective regulation of IRESdependent translation. We also propose a speculative model that suggests that the dynamics of pre-assembly and nuclear import of H/ACA RNPs are crucial regulatory steps that can be finely controlled in the cytoplasm in response to developmental, differentiative and stress stimuli. [ABSTRACT FROM AUTHOR]

Biological membranes consist of fluid bilayers with many lipid and protein components. This fluidity implies a high flexibility that allows the membranes to attain a large variety of different shapes. One important shape parameter is the spontaneous curvature, which describes the asymmetry between the two leaflets of a bilayer and can be changed by adsorption of 'particles' such as ions or proteins from the aqueous phases. Membrane fluidity also implies that the membranes can change their local composition via lateral diffusion and form intramembrane compartments. Two mechanisms for the formation of such compartments can be distinguished: membrane segmentation arising from structured environments and domain formation as a result of phase separation within the membranes. The interplay between these two mechanisms provides a simple and generic explanation for the difficulty to observe phase domains in vivo. Intramembrane domains can form new membrane compartments via budding and tubulation processes. Which of these two processes actually occurs depends on the fluid-elastic properties of the domains, on the adsorption kinetics, and on external constraints arising, e.g., from the osmotic conditions. Vesicles are predicted to unbind from adhesive surfaces via tubulation when the spontaneous curvature of their membranes exceeds a certain threshold value. [ABSTRACT FROM AUTHOR]

Ara h 8 is the peanut allergen homologous to the birch pollen allergen Bet v 1. Because Bet v 1 has been shown to bind lipophilic ligands, the aim of this investigation was to determine the impact of lipid binding and roasting on the Ara h 8 structure and their influences on allergenicity. For the characterization of natural Ara h 8 (nAra h 8) from roasted and unroasted peanuts, circular dichroism spectroscopy, hydrophobic binding assay, immunohistochemistry, and immunoblot with sera of peanut allergic patients were performed and compared with results from recombinant Ara h 8 (rAra h 8) and Bet v 1. rAra h 8 displayed stronger hydrophobicity than rBet v 1. Patients' sera showed IgE reactivity with rAra h 8 and nAra h 8 from roasted peanuts, whereas fewer sera recognized nAra h 8 from unroasted peanuts. Simulated gastric digestion experiments demonstrated low proteolytic stability of rAra h 8, whereas the stability of nAra h 8 was increasingly higher in unroasted and roasted peanuts. The results demonstrate that IgE reactivity and thermal and proteolytic stability are reinforced in nAra h 8 after roasting, most likely due to Maillard reactions, lipid oxidations, and lipophilic associations. These aspects must be considered when estimating the allergenicity of Bet v 1-homologous proteins. [ABSTRACT FROM AUTHOR]

RNA helicases are a diverse class of enzymes that catalyze the ATP-dependent unwinding of RNA duplex regions. Donsbach and Klostermeier review the general regulatory principles that govern RNA helicase activity, and illustrate these principles with selected examples from different RNA helicase families ([5]). RNA helicases have also been associated with the regulation of liquid-liquid phase separation of RNA and RNA-protein complexes in the cell. [Extracted from the article]

The diversity of cellular actin functions is attained by the activation of actin nucleator complexes, which initiate the polymerization of actin monomers into a helical double-stranded filament at defined subcellular compartments. Next to actin functions at the cell membrane, including different forms of membrane protrusions and invaginations, actin dynamics at intracellular membranes has recently become a research focus. Experiments addressing the vesicle-associated Spir WH2 domain containing actin nucleators have provided novel mechanistic insights into the function of actin dynamics at intracellular membranes. Spir proteins are targeted by a modified FYVE zinc finger motif toward endosomal and vesicle membranes, where they interact and cooperate with the distinct nucleators of the FMN subfamily of formins in the nucleation of actin filaments. The function of the Spir/formin actin nucleator complex is closely related to the Rab11 small G protein, which is a key regulator of recycling and exocytic transport processes. Together with the actin motor protein and Rab11 effector myosin Vb, Spir/formin nucleated actin filaments mediate actin-dependent vesicle transport processes. Drosophila and mouse genetic studies as well as cell biology experiments point toward an important role of the Spir/formin complex in oocyte maturation and in the structure and signaling of the nervous system. [ABSTRACT FROM AUTHOR]

Neural precursor cells (NPCs) are lineage-restricted neural stem cells with limited self-renewal, giving rise to a broad range of neural cell types such as neurons, astrocytes, and oligodendrocytes. Despite this developmental potential, the differentiation capacity of NPCs has been controversially discussed concerning the trespassing lineage boundaries, for instance resulting in hematopoietic competence. Assessing their in vitro plasticity, we isolated nestin+/Sox2+, NPCs from the adult murine hippocampus. In vitro-expanded adult NPCs were able to form neurospheres, self-renew, and differentiate into neuronal, astrocytic, and oligodendrocytic cells. Although NPCs cultivated in early passage efficiently gave rise to neuronal cells in a directed differentiation assay, extensively cultivated NPCs revealed reduced potential for ectodermal differentiation. We further observed successful differentiation of long-term cultured NPCs into osteogenic and adipogenic cell types, suggesting that NPCs underwent a fate switch during culture. NPCs cultivated for more than 12 passages were aneuploid (abnormal chromosome numbers such as 70 chromosomes). Furthermore, they showed growth factor-independent proliferation, a hallmark of tumorigenic transformation. In conclusion, our findings substantiate the lineage restriction of NPCs from adult mammalian hippocampus. Prolonged cultivation results, however, in enhanced differentiation potential, which may be attributed to transformation events leading to aneuploid cells. [ABSTRACT FROM AUTHOR]

Overwhelming evidence shows that oxidative stress is a major cause in development of brain disorders. Low activity of the reactive oxygen species (ROS)-degrading system as well as high levels of oxidative damage markers have been observed in brain tissue of patients with neurodegenerative and other brain diseases to a larger extent than in healthy individuals. Many studies aimed to develop effective and safe antioxidant strategies for the therapy or prevention of brain diseases. Nevertheless, it became clear that rigorous suppression of ROS is deleterious for normal cell functioning. Thus, approaches that can regulate the ROS levels over a wide range, from inhibition to induction, will be a powerful tool for neuroprotection. A most prominent target for such ROS management is the family of peroxisome proliferator-activated receptors (PPARs). All three members (PPAR-α, -β/δ and -γ) of this nuclear receptor subfamily form a tightly connected triad. For individual PPAR isoforms, neuroprotective properties have been well proven. Their involvement in regulation of ROS production and degradation underlies the therapeutic effects. Nevertheless, the current paradigms of the involvement of PPAR in neuroprotective therapy ignore such interconnections of PPARs and aim at antioxidant effects of individual PPAR isoforms, but do not take into account the necessity of careful regulation of ROS levels. The present review (i) summarizes the data, which support the concept of the PPAR triad in brain, (ii) demonstrates that usage of the PPAR triad allows the regulation of PPAR-dependent genes over a wide range, from inhibition to upregulation, and (iii) summarizes the known data concerning the PPAR triad involvement in regulation of ROS. Our report opens new directions in the field of PPAR/ROS-related neuroscience research. [ABSTRACT FROM AUTHOR]

The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans. All A3 proteins can deaminate cytidines to uridines in single-stranded DNA (ssDNA), generated during viral reverse transcription. A3 proteins have either one or two cytidine deaminase domains (CD). The CDs coordinate a zinc ion, and their amino acid specificity classifies the A3s into A3Z1, A3Z2, and A3Z3. A3 proteins occur as monomers, dimers, and large oligomeric complexes. Studies on the nature of A3 oligomerization, as well as the mode of interaction of A3s with RNA and ssDNA are partially controversial. High-resolution structures of the catalytic CD2 of A3G and A3F as well as of the single CD proteins A3A and A3C have been published recently. The NMR and X-ray crystal structures show globular proteins with six α-helices and five β sheets arranged in a characteristic motif (α1-β1-β2/2′-α2-β3-α3-β4-α4-β5-α5-α6). However, the detailed arrangement and extension of individual structure elements and their relevance for A3 complex formation and activity remains a matter of debate and will be highlighted in this review. [ABSTRACT FROM AUTHOR]

Oxazolidinone antibiotics bind to the highly conserved peptidyl transferase center in the ribosome. For developing selective antibiotics, a profound understanding of the selectivity determinants is required. We have performed for the first time technically challenging molecular dynamics simulations in combination with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations of the oxazolidinones linezolid and radezolid bound to the large ribosomal subunits of the eubacterium Deinococcus radiodurans and the archaeon Haloarcula marismortui. A remarkably good agreement of the computed relative binding free energy with selectivity data available from experiment for linezolid is found. On an atomic level, the analyses reveal an intricate interplay of structural, energetic, and dynamic determinants of the species selectivity of oxazolidinone antibiotics: A structural decomposition of free energy components identifies influences that originate from first and second shell nucleotides of the binding sites and lead to (opposing) contributions from interaction energies, solvation, and entropic factors. These findings add another layer of complexity to the current knowledge on structure-activity relationships of oxazolidinones binding to the ribosome and suggest that selectivity analyses solely based on structural information and qualitative arguments on interactions may not reach far enough. The computational analyses presented here should be of sufficient accuracy to fill this gap. [ABSTRACT FROM AUTHOR]

In the last two decades a great deal of evidence has been collected on the key role of endothelial progenitor cells (EPC) in the mechanisms of vascular healing. The role of EPC as a marker of vascular health and prognosis of cardiovascular disease is already consolidated. This review aims to examine and evaluate recent data regarding EPC, as biomarkers, prognostic factor and potential therapy in cardiovascular disease. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter. [ABSTRACT FROM AUTHOR]

Differentiation of human mesenchymal stem cells (MSCs) to metabolically active hepatocytes depends on different regulatory factors. Trans-differentiation of stem cells into specific cell lineage in the presence of specific stimuli is associated with the molecular and cellular damage. The aim of the present study was to examine the role of P53 in the regulation of cyclooxygenase-2 (COX-2) expression and the generation of protein and lipid oxidation during trans-differentiation of MSCs into hepatocyte-like cells. During the 3-week differentiation process of MSCs to hepatocyte-like cells we found that expression liver-specific markers was associated with increased levels of lipid peroxidation and protein carbonyl formation. Expression of P53 and COX-2 at mRNA and protein levels were evaluated in MSCs before and after differentiation on days 7, 14 and 21. We showed that the up-regulation of COX-2 was associated with augmentation of the rate of cell proliferation, morphological and biochemical changes of hepatocytes-like cells. However, in parallel the P53 at the mRNA level was down-regulated, and at protein levels accumulation in the nuclei was reduced during the hepatogenic differentiation time. Our results may suggest a P53-COX-2 pathway in the regulation of hepatogenic differentiation of stem cells, which is linked to differentiation-dependent molecular oxidative damage. [ABSTRACT FROM AUTHOR]

The pleiotropic physiological functions of interleukin (IL-)6 type cytokines range from embryonic development and tissue homoeostasis to neuronal development and T cell differentiation. In contrast, imbalance of the well-controlled cytokine signaling network leads to chronic inflammatory diseases and cancer. IL-6 and IL-11 both signal through a homodimer of the ubiquitously expressed β-receptor glycoprotein 130 (gp130). Specificity is gained through an individual IL-6/IL-11 α-receptor, which does not directly participate in signal transduction, although the initial cytokine binding event to the α-receptor leads to the final complex formation with the β-receptors. Both cytokines activate the same downstream signaling pathways, which are predominantly the mitogen-activated protein kinase (MAPK)-cascade and the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. However, recent studies have highlighted divergent roles of the two related cytokines. Here, we will discuss how the biochemical similarities are translated into unique and non-redundant functions of IL-6 and IL-11 in vivo and illustrate strategies for cytokine-specific therapeutic intervention. [ABSTRACT FROM AUTHOR]

Maackia amurensis agglutinin is a NeuNAcα (2-3) Galβ (1-4) GlcNAc/Glc-specific lectin, which was shown to have diagnostic potential in cancers of different origin. In a previous report, we demonstrated that GM3 specific IgG from bronchoalveolar lavage fluid (BALF) of non-small cell lung cancer (NSCLC) patients interacted with ∼66kDa membrane glycoprotein band of NSCLC cell lines, which was also recognised by this lectin. This observation prompted us to assess the potential of Maackia amurensis agglutinin in NSCLC. Accordingly, we examined the reactivity of this lectin with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. Maackia amurensis agglutinin showed strong reactivity, specifically with cells and biopsy samples of NSCLC origin. Furthermore, this lectin was found to induce apoptosis in NSCLC cells. The mechanism of this lectin-induced apoptosis involved downregulation of Bcl-XL, upregulation of Bax, release of cytochrome c and activation of procaspase-3. Collectively our results have suggested that Maackia amurensis agglutinin may have the potential to serve as a unique probe for detection of NSCLC and also as a specific apoptosis-inducing agent in NSCLC cells. [ABSTRACT FROM AUTHOR]

A role for the kinin B1 receptor in energy-homeostatic processes was implicated in previous studies; notably, the studies where kinin B1 receptor knockout mice (B1-/-) were shown to have impaired adiposity, impaired leptin and insulin production, lower feed efficiency, protection from liver steatosis and diet-induced obesity when fed a high fat diet (HFD). In particular, in a model where the B1 receptor is expressed exclusively in the adipose tissue, it rescues the plasma insulin concentration and the weight gain seen in wild type mice. Taking into consideration that leptin participates in the formation of hypothalamic nuclei, which modulate energy expenditure, and feeding behavior, we hypothesized that these brain regions could also be altered in B1-/- mice. We observed for the first time a difference in the gene expression pattern of cocaine and amphetamine related transcript (CART) in the (lateral hypothalamic area (LHA) resulting from the deletion of the kinin B1 receptor gene. The correlation between CART expression in the LHA and the thwarting of diet-induced obesity corroborates independent correlations between CART and obesity. Furthermore, it seems to indicate that the mechanism underlying the 'lean' phenotype of B1-/- mice does not stem solely from changes in peripheral tissues but may also receive contributions from changes in the hypothalamic machinery involved in energy homeostasis processes. [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding glucocerebrosidase ( GBA1) cause Gaucher disease (GD), a lysosomal storage disease with recessive inheritance. Glucocerebrosidase (GCase) is a lysosomal lipid hydrolase that digests glycolipid substrates, such as glucosylceramide and glucosylsphingosine. GBA1 mutations have been implicated in Lewy body diseases (LBDs), such as Parkinson's disease and dementia with Lewy bodies. Parkinsonism occurs more frequently in certain types of GD, and GBA1 mutation carriers are more likely to have LBDs than non-carriers. Furthermore, GCase is often found in Lewy bodies, which are composed of α-synuclein fibrils as well as a variety of proteins and vesicles. In this review, we discuss potential mechanisms of action of GBA1 mutations in LBDs with particular emphasis on α-synuclein aggregation by reviewing the current literature on the role of GCase in lysosomal functions and glycolipid metabolism. [ABSTRACT FROM AUTHOR]

This review takes a comparative look at the various scenarios where ribosomes are degraded in bacteria and eukaryotes with emphasis on studies involving Escherichia coli and Saccharomyces cerevisiae. While the molecular mechanisms of degradation in bacteria and yeast appear somewhat different, we argue that the underlying causes of ribosome degradation are remarkably similar. In both model organisms during ribosomal assembly, partially formed pre-ribosomal particles can be degraded by at least two different sequentially-acting quality control pathways and fully assembled but functionally faulty ribosomes can be degraded in a separate quality control pathway. In addition, ribosomes that are both structurally- and functionally-sound can be degraded as an adaptive measure to stress. [ABSTRACT FROM AUTHOR]

To look for oral proteasome inhibitors, daily injested food is the best source for cancer chemoprevention. A combination of active components from vegetables, coffee, tea, and fruit could be more efficient to inhibit 26S proteasome activities for preventing cancer diseases. Tannic acid and quercetin have been shown to strongly inhibit 26S proteasome activity, but the molecular target involved remains unknown. Overlay assay, peptide assay, Western blot, and 2-D gels were used to assess the combination of quercetin and tannic acid as a potential inhibitor. Here, we demonstrated that the combination of quercetin and tannic acid (1) synergistically suppresses chymotrypsin-, caspase-, and trypsin-like proteolytic activities, (2) are tightly binding substrates, (3) do not perturb the proteasome structure, (4) inhibit the 26S proteasome affected by ubiquitin, ATP, or β-casein, and (5) inhibit β-casein degradation by the 26S proteasome in vitro. Finally, the inhibition of the proteasome by a combination of quercetin plus tannic acid in Hep-2 cells resulted in the induction of S5a at low dose, accumulation of ubiquitin, and the cleavage of pro-caspase-3, followed by the induction of apoptotic cell death. Evaluating the combination of quercetin and tannic acid as an oral drug to prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this combination. [ABSTRACT FROM AUTHOR]

Somatic angiotensin I-converting enzyme (ACE) has two homologous active sites (N and C domains) that show differences in various biochemical properties. In a previous study, we described the use of positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain (Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P3 to P1′ positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz= ortho-aminobenzoic acid; Dnp=2,4-dinitrophenyl) showed the highest selectivity for ACE N-domain ( k cat /K m=1.76 μ m-1·s-1). Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable. [ABSTRACT FROM AUTHOR]

During the EUROCarbDB project our group developed the GlycanBuilder and GlycoWorkbench glycoinformatics tools. This short communication summarizes the capabilities of these two tools and updates which have been made since the original publications in 2007 and 2008. GlycanBuilder is a tool that allows for the fast and intuitive drawing of glycan structures; this tool can be used standalone, embedded in web pages and can also be integrated into other programs. GlycoWorkbench has been designed to semi-automatically annotate glycomics data. This tool can be used to annotate mass spectrometry (MS) and MS/MS spectra of free oligosaccharides, N and O-linked glycans, GAGs (lycosminolycans) and glycolipids, as well as MS spectra of glycoproteins. [ABSTRACT FROM AUTHOR]

The members of the YidC/Oxa1/Alb3 protein family are evolutionary conserved in all three domains of life. They facilitate the insertion of membrane proteins into bacterial, mitochondrial, and thylakoid membranes and have been implicated in membrane protein folding and complex formation. The major classes of substrates are small hydrophobic subunits of large energy-transducing complexes involved in respiration and light capturing. All YidC-like proteins share a conserved membrane region, whereas the N- and C-terminal regions are diverse and fulfill accessory functions in protein targeting. [ABSTRACT FROM AUTHOR]

Chloroplast biogenesis often requires a tight orchestration between gene expression (both plastidial and nuclear) and translocation of ∼3000 nuclear-encoded proteins into the organelle. Protein translocation is achieved via two multimeric import machineries at the outer (TOC) and inner (TIC) envelope of chloroplast, respectively. Three components constitute the core element of the TOC complex: a β-barrel protein translocation channel Toc75 and two receptor constituents, Toc159 and Toc34. A diverse set of distinct TOC complexes have recently been characterized and these diversified TOC complexes have evolved to coordinate the translocation of differentially expressed proteins. This review aims to describe the recent discoveries relating to the typical characteristics of these distinct TOC complexes, particularly the receptor constituents, which are the main contributors for TOC complex diversification. [ABSTRACT FROM AUTHOR]

The Wnt/β-catenin and the Ras/mitogen-activated protein kinase (MAPK) pathways play important roles in cancer development. Both pathways have been studied discretely, but the mechanisms of possible crosstalk are still not fully understood. We have previously shown that β-catenin and MAPK signaling interfere with each other in murine liver in vivo and in vitro. Here, we show that dual specificity phosphatases (Dusps) 6 and 14, known to play an essential role in regulating MAPK pathway activity via feedback mechanisms, are up-regulated by activation of β-catenin in murine liver cells, whereas the epidermal growth factor receptor, an upstream effector in the Ras/MAPK cascade, is down-regulated by β-catenin. In addition, we identified a β-catenin-binding site within the Dusp6 promoter, which is responsible for the activation of the promoter by β-catenin signaling, and demonstrated reduced inducibility of MAPK signaling in cultured mouse hepatoma cells following β-catenin activation. Thus, β-catenin is able to inhibit activation of the Egfr/Ras/MAPK signaling cascade, both at the receptor level and by interfering with MAPK activity via Dusps. [ABSTRACT FROM AUTHOR]

Phytopathogen transcription activator-like effectors (TALEs) bind DNA in a sequence specific manner in order to manipulate host transcription. TALE specificity correlates with repeat variable diresidues in otherwise highly stereotypical 34-35mer repeats. Recently, the crystal structures of two TALE DNA-binding domains have illustrated the molecular basis of the TALE cipher. The structures show that the TALE repeats form a right-handed superhelix that is wound around largely undistorted B-DNA to match its helical parameters. Surprisingly, repeat variable residue 1 is not in contact with the bases. Instead, it is involved in hydrogen bonding interactions that stabilize the overall structure of the protein. Repeat variable residue 2 contacts the top strand base and forms sequence-specific hydrogen bonds and/or van der Waals contacts. Very unexpectedly, bottom strand bases are exposed to solvent and do not make any direct contacts with the protein. This review contains a summary of TALE biology and applications and a detailed description of the recent breakthroughs that have provided insights into the molecular basis of the TALE code. [ABSTRACT FROM AUTHOR]

The 20S proteasome core particle (CP) is the proteolytically active key element of the ubiquitin proteasome system that directs the majority of intracellular protein degradation in eukaryotic cells. Over the past decade, the CP has emerged as an anticancer therapy target after approval of the first-in-class drug bortezomib (Velcade®) by the US Food and Drug Administration. However, bortezomib and all second-generation CP inhibitors that are currently explored in clinical phase studies react covalently and most often irreversibly with the proteolytic sites of the CP, hereby causing permanent CP blockage. Furthermore, reactive head groups result in unspecific binding to proteasomal active centers and in substantial enzymatic off-target activities that translate to severe side effects. Thus, reversible proteasome inhibitors might be a promising alternative, overcoming these drawbacks, but are challenging with respect to their urge for thorough enthalpic and entropic optimization. This review describes developments in the hitherto neglected field of reversible proteasome inhibitors focusing on insights gained from crystal structures, which provide valuable knowledge and strategies for future directions in drug development. [ABSTRACT FROM AUTHOR]