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FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFPFKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo. The identified NLS signal is located in a basic domain. Detailed mutagenesis studies revealed that residues K188 and K191 are crucial for the nuclear targeting of FKBP39 and its nucleoplasmin-like (NPL) domain contains the sequence that controls the nucleolar-specific translocation of the protein. These results show that FKBP39 possesses a specific NLS in close proximity to a putative helix-turn-helix (HTH) motif and FKBP39 may bind DNA in vivo and in vitro. [ABSTRACT FROM AUTHOR]

We investigated thoroughly the effect of lncRNA PART1 on prostate cancer cells proliferation and apoptosis, through regulating toll-like receptor (TLR) pathways. LncRNA PART1 expression was also examined by quantitative real-time polymerase chain reactions (qRT-PCR) in human tissues and the cells lines LNCaP and PC3. After transfection with si-PART1 or control constructs, the cell viability was measured by MTS and colony formation assays. In addition, the apoptosis rate of the prostate cancer cells was validated by TUNEL staining. Relationships between lncRNA PART1 expression and TLR pathway genes were demonstrated by qRT-PCR and Western blotting. High levels of lncRNA PART1 expression were correlated with advanced cancer stage and predication of poor survival. LncRNA PART1 levels was increased in PCa cells treated with 5a-dihydrotestosterone (DHT), confirming PART1 was directly induced by androgen. Moreover, down-regulation of lncRNA PART1 inhibited prostate cancer cell proliferation and accelerated cell apoptosis. In addition, lncRNA PART1 induced downstream genes expression in TLR pathways including TLR3, TNFSF10 and CXCL13 to further influence prostate cancer cells, indicating its carcinogenesis on prostate cancer. LncRNA PART1 promoted cell proliferation ability and apoptosis via the inhibition of TLR pathways in prostate cancer. LncRNA PART1 could hence be considered as a new target in the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

Metformin is a widely used antidiabetic drug, and there is evidence among diabetic patients that metformin is a chemopreventive agent against multiple cancers. There is also evidence in human studies that metformin is a cancer chemotherapeutic agent, and several clinical trials that use metformin alone or in combination with other drugs are ongoing. In vivo and in vitro cancer cell culture studies demonstrate that metformin induces both AMPK-dependent and AMPK-independent genes/pathways that result in inhibition of cancer cell growth and migration and induction of apoptosis. The effects of metformin in cancer cells resemble the patterns observed after treatment with drugs that downregulate specificity protein 1 (Sp1), Sp3 and Sp4 or by knockdown of Sp1, Sp3 and Sp4 by RNA interference. Studies in pancreatic cancer cells clearly demonstrate that metformin decreases expression of Sp1, Sp3, Sp4 and pro-oncogenic Sp-regulated genes, demonstrating that one of the underlying mechanisms of action of metformin as an anticancer agent involves targeting of Sp transcription factors. These observations are consistent with metformin-mediated effects on genes/pathways in many other tumor types. [ABSTRACT FROM AUTHOR]

We describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on live Escherichia coli cells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as the target, Anticalins were selected from a naïve bacterial library. The Anticalin with the highest affinity (KD = 13 nm), as determined towards recombinant full-length Hsp70 by real-time surface plasmon resonance analysis, was improved to KD = 510 pm by doped random mutagenesis and another cycle of E. coli surface display, followed by rational combination of mutations. This Anticalin, which recognises a linear peptide epitope located in the interdomain linker of Hsp70, was demonstrated to specifically bind Hsp70 in its membrane-associated form in immunofluorescence microscopy and via flow cytometry using the FaDu cell line, which is positive for surface Hsp70. The radiolabelled and PASylated Anticalin revealed specific tumour accumulation in xenograft mice using positron emission tomography (PET) imaging. Furthermore, after enzymatic coupling to the protein toxin gelonin, the Anticalin showed potent cytotoxicity on FaDu cells in vitro. [ABSTRACT FROM AUTHOR]

In 2012, we briefly summarized the mechanisms, pathophysiological roles and methods for analyzing mitophagy. As then, the mitophagy field has continued to grow rapidly, and many new molecular mechanisms regulating mitophagy and molecular tools for monitoring mitophagy have been discovered and developed. Therefore, the purpose of this review is to update information regarding these advances in mitophagy while focusing on basic molecular mechanisms of mitophagy in different organisms and its pathophysiological roles. We also discuss the advantage and limitations of current methods to monitor and quantify mitophagy in cultured cells and in vivo mouse tissues. [ABSTRACT FROM AUTHOR]

The article announces that Vanessa Ferreira Merino is honored with the Paper of the Year 2005 Award by the Editors of Biological Chemistry and the German Society for Biochemistry and Molecular Biology for her paper on "Molecular structure and transcriptional regulation by nuclear factor-κB of the mouse kinin B1 receptor gene," that was published in June 2005 issue of Biological Chemistry. A brief profile of Vanessa with a discussion on her paper is provided.

Features Ying Wang and Iris Lorenzo, recipients of the 2004 Paper of the Year Award for their study that described how metal-responsive transcription factor MTF-1 selects the types of zinc-dependent metal response elements. Profile of Wang and Lorenzo; Overview of their study.

Many studies suggest that BACE 1 is the genuine β-secretase; however, this is not undisputed. The wild-type (WT) β-site of the amyloid precursor protein (APP) present in the worldwide population is cleaved very slowly ( kcat/ Km: approx. 50 -1 s-1), while proteases acting on relevant substrates are much more efficient ( kcat/ Km: 104–106 -1 s-1). Knock-out of BACE 1 in mouse markedly reduces Aβ formation. Nevertheless, studies in other systems show that knock-out experiments in rodents and corresponding genetic defects in human may reveal different phenotypes. Considering these issues, we searched for other β-secretase candidate(s), identified cathepsin D, and evaluated properties of cathepsin D related to BACE 1 that were not examined previously. The kinetic constants ( kcat, Km, kcat/ Km) for cleaving peptides with β-sites of the WT or the mutated Swedish families (SW) APP by human BACE 1 and cathepsin D were determined and found to be similar. Western blots reveal that in human brain cathepsin D is approximately 280-fold more abundant than BACE 1. Furthermore, pepstatin A strongly inhibits the cleavage of SW and WT peptides by both brain extracts and cathepsin D, but not by BACE 1. These findings indicate that β-secretase activity observed in brain extracts is mainly due to cathepsin D. Nevertheless, as both BACE 1 and cathepsin D show poor activity towards the WT β-site sequence, it is necessary to continue the search for additional β-secretase candidate(s). [ABSTRACT FROM AUTHOR]

Decades of chemical, biochemical and pathophysiological research have established the relevance of post-translational protein modifications induced by processes related to oxidative stress, with critical reflections on cellular signal transduction pathways. A great deal of the so-called 'redox regulation' of cell function is in fact mediated through reactions promoted by reactive oxygen and nitrogen species on more or less specific aminoacid residues in proteins, at various levels within the cell machinery. Modifications involving cysteine residues have received most attention, due to the critical roles they play in determining the structure/function correlates in proteins. The peculiar reactivity of these residues results in two major classes of modifications, with incorporation of NO moieties (S-nitrosation, leading to formation of protein S-nitrosothiols) or binding of low molecular weight thiols (S-thionylation, i.e. in particular S-glutathionylation, S-cysteinylglycinylation and S-cysteinylation). A wide array of proteins have been thus analyzed in detail as far as their susceptibility to either modification or both, and the resulting functional changes have been described in a number of experimental settings. The present review aims to provide an update of available knowledge in the field, with a special focus on the respective (sometimes competing and antagonistic) roles played by protein S-nitrosations and S-thionylations in biochemical and cellular processes specifically pertaining to pathogenesis of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

OmpF porin from the outer membrane of Yersinia pseudotuberculosis was cloned into pET-40b(+) plasmid. Using E. coli Rosetta (DE3) strain, MX medium, IPTG concentration of 0.2 mm and post-induction cultivation at 14°C overnight allowed us to obtain a water-soluble form of the recombinant protein (rs-OmpF). Rs-OmpF was shown to have the ordered spatial structure at the levels of secondary and tertiary structure. Rs-OmpF was found to be effective as diagnostic antigen in ELISA for pseudotuberculosis diagnostics. [ABSTRACT FROM AUTHOR]

The efficacy of HIV-1 protease (PR) inhibition therapies is often compromised by the emergence of mutations in the PR molecule that reduces the binding affinity of inhibitors while maintaining viable catalytic activity and affinity for natural substrates. In the present study, we used a recombinant HIV-1 C-SA PR and a recently reported variant for inhibition (Ki, IC50) and thermodynamic studies against nine clinically used inhibitors. This is the first time that binding free energies for C-SA PR and the mutant are reported. This variant PR harbours a mutation and insertion (I36T↑T) at position 36 of the C-SA HIV-1 PR, and did not show a significant difference in the catalytic effect of the HIV-1 PR. However, the nine clinically approved HIV PR drugs used in this study demonstrated weaker inhibition and lower binding affinities toward the variant when compared to the wild type HIV-1 PR. All the protease inhibitors (PIs), except Amprenavir and Ritonavir exhibited a significant decrease in binding affinity (p < 0.0001). Darunavir and Nelfinavir exhibited the weakest binding affinity, 155- and 95-fold decreases respectively, toward the variant. Vitality values for the variant PR, against the seven selected PIs, confirm the impact of the mutation and insertion on the South African HIV-1 subtype C PR. This information has important clinical implications for thousands of patients in Sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

It has been more than 10 years that the first prophylactic papillomavirus vaccine became available, although distribution has been mainly limited to the more affluent countries. The first two vaccines have been a great success, hundreds of millions of women and a much smaller number of men have been vaccinated ever since. In a few countries with high vaccination coverage, in particular Australia but also parts of Great Britain and others, clinical impact of vaccination programs is already visible and there are indications for herd immunity as well. Vaccine efficacy is higher than originally estimated and the vaccines have an excellent safety profile. Gardasil9 is a second generation HPV virus-like particle vaccine that was licensed in 2015 and there are more to come in the near future. Currently, burning questions in respect to HPV vaccination are the duration of protection -- especially in regard to cross-protection -- reduction of the three-dose regimen and its impact on cross-protection; and duration of response, as well as protection against oropharyngeal HPV infections. Furthermore, researchers are seeking to overcome limitations of the VLP vaccines, namely low thermal stability, cost, invasive administration, limited coverage of non-vaccine HPV types, and lack of therapeutic efficacy. In this review we summarize the current status of licensed VLP vaccines and address questions related to second and third generation HPV vaccines. [ABSTRACT FROM AUTHOR]

Accordingly, membrane transporter dysfunctions often result in severe disease conditions and many membrane transporters are established drug targets for pharmacological disease control. Among the class of membrane proteins, membrane transporters are essential regulatory molecules that allow the passage of mostly membrane-impermeable molecules across biomembranes. As a common feature, most of them share the presence of alpha-helical transmembrane domains traversing the membrane, with the notable exception of beta-barrel proteins in the outer membranes of gram-negative bacteria and mitochondria. [Extracted from the article]

Alpha-synuclein (α-syn) is an abundant neuronal protein whose physiological function, even if still not completely understood, has been consistently related to synaptic function and vesicle trafficking. A group of disorders known as synucleinopathies, among which Parkinson's disease (PD), is deeply associated with the misfolding and aggregation of α-syn, which can give rise to proteinaceous inclusion known as Lewy bodies (LB). Proteostasis stress is a relevant aspect in these diseases and, currently, the presence of oligomeric α-syn species rather than insoluble aggregated forms, appeared to be associated with cytotoxicity. Many observations suggest that α-syn is responsible for neurodegeneration by interfering with multiple signaling pathways. α-syn protein can directly form plasma membrane channels or modify with their activity, thus altering membrane permeability to ions, abnormally associate with mitochondria and cause mitochondrial dysfunction (i.e. mitochondrial depolarization, Ca2+ dys-homeostasis, cytochrome c release) and interfere with autophagy regulation. The picture is further complicated by the fact that single point mutations, duplications and triplication in α-syn gene are linked to autosomal dominant forms of PD. In this review we discuss the multi-faced aspect of α-syn biology and address the main hypothesis at the basis of its involvement in neuronal degeneration. [ABSTRACT FROM AUTHOR]

Interleukin-1β (IL-1β) is a cytokine protein expressed by cardiomyocyte in myocardial damage-associated inflammation. Although several methods are currently available for treatment of heart diseases none of them are completely successful. Recently, stem cells have gained enormous attention and are expected to play a significant role for treating heart diseases. 5-Azacytidine (5-aza) has recently been found to cause stimulation of stem cells to differentiate into cardiomyocytes. Here we present the determination of whether IL-1β can induce the differentiation of mesenchymal stem cells (MSCs) to cardiomyocytes. MSCs were derived from bone marrow, propagated and then cultured in differentiation medium supplemented with 5-aza and IL-1β (at two levels, 5 and 10 ng/ml). After 21 days, total RNA was extracted and cDNA synthesis was carried out. Quantitative polymerase chain reaction (Q-PCR) was performed for detecting cardiac-specific markers. Besides, to investigate the expression of cardiac markers in protein levels, immunocytochemistry was done by specific antibodies. Ultimately, cardiac markers expression suggested that IL-1β and 5-aza synergistically induces the cardiomyocyte differentiation. [ABSTRACT FROM AUTHOR]

The CARMA1-BCL10-MALT1 (CBM) signalosome triggers canonical NF-?B signaling and lymphocyte activation upon antigen-receptor stimulation. Genetic studies in mice and the analysis of human immune pathologies unveiled a critical role of the CBM complex in adaptive immune responses. Great progress has been made in elucidating the fundamental mechanisms that dictate CBM assembly and disassembly. By bridging proximal antigenreceptor signaling to downstream signaling pathways, the CBM complex exerts a crucial scaffolding function. Moreover, the MALT1 subunit confers a unique proteolytic activity that is key for lymphocyte activation. Deregulated 'chronic' CBM signaling drives constitutive NF-κB signaling and MALT1 activation, which contribute to the development of autoimmune and inflammatory diseases as well as lymphomagenesis. Thus, the processes that govern CBM activation and function are promising targets for the treatment of immune disorders. Here, we summarize the current knowledge on the functions and mechanisms of CBM signaling in lymphocytes and how CBM deregulations contribute to aberrant signaling in malignant lymphomas. [ABSTRACT FROM AUTHOR]

Kallikrein-related peptidase-3 (KLK3, known also as prostate-specific antigen, PSA) is highly expressed in the prostate. KLK3 possess antiangiogenic activity, which we have found to be related to its proteolytic activity. Thus, it may be possible to slow down the growth of prostatic tumors by enhancing this activity. We have developed peptides that enhance the proteolytic activity of KLK3. As these peptides are degraded in circulation and rapidly excreted, we have started to modify them and have succeeded in creating bioactive and more stable pseudopeptides. We have also identified small molecules stimulating the activity of KLK3, especially in synergy with peptides. [ABSTRACT FROM AUTHOR]

Inflammation is essential to protect the host from exogenous and endogenous dangers that ultimately lead to tissue injury. The consequent tissue repair is intimately associated with the fate of the inflammatory response. Restoration of tissue homeostasis is achieved through a balance between pro-inflammatory and antiinflammatory/ pro-resolving mediators. In chronic inflammatory diseases such balance is compromised, resulting in persistent inflammation and impaired healing. During the last two decades the glucocorticoid-regulated protein Annexin A1 (AnxA1) has emerged as a potent proresolving mediator acting on several facets of the innate immune system. Here, we review the therapeutic effects of AnxA1 on tissue healing and repairing together with the molecular targets responsible for these complex biological properties. [ABSTRACT FROM AUTHOR]

The enzyme members of the dipeptidyl peptidase 4 (DPP4) gene family have the very unusual capacity to cleave the post-proline bond to release dipeptides from the N-terminus of peptide/protein substrates. DPP4 and related enzymes are current and potential therapeutic targets in the treatment of type II diabetes, inflammatory conditions and cancer. Despite this, the precise biological function of individual dipeptidyl peptidases (DPPs), other than DPP4, and knowledge of their in vivo substrates remains largely unknown. For many years, identification of physiological DPP substrates has been difficult due to limitations in the available tools. Now, with advances in mass spectrometry based approaches, we can discover DPP substrates on a system wide-scale. Application of these approaches has helped reveal some of the in vivo natural substrates of DPP8 and DPP9 and their unique biological roles. In this review, we provide a general overview of some tools and approaches available for protease substrate discovery and their applicability to the DPPs with a specific focus on DPP9 substrates. This review provides comment upon potential approaches for future substrate elucidation. [ABSTRACT FROM AUTHOR]

I was given the honor of delivering the 2015 Lifetime Membership Award lecture at the International Proteolysis Society's annual meeting held in Penang, Malaysia in October 2015. It gave me an opportunity to look back on how I started my research on matrix metalloproteinases (MMPs) and how I continued to work on these proteinases for the next 42 years. This is a series of sketches from the personal journey that I took with MMPs, starting from the purification of metalloproteinases, cloning, structural studies, then to a more recent encounter, endocytic regulation of matrix-degrading metalloproteinases. [ABSTRACT FROM AUTHOR]

We received early access to the newest releases of exome sequencing products, namely Agilent SureSelect v6 (Agilent, Santa Clara, CA, USA) and NimbleGen MedExome (Roche NimbleGen, Basel, Switzerland), and we conducted whole exome sequencing (WES) of several DNA samples with each of these products in order to assess their performance. Here, we provide a detailed evaluation of the original, normalized (with respect to the different target sizes), and trimmed data sets and compare them in terms of the amount of duplicates, the reads on target, and the enrichment evenness. In addition to these general statistics, we performed a detailed analysis of the frequently mutated and newly described genes found in 'The Deciphering Developmental Disorders Study' published very recently (Fitzgerald, T.W., Gerety, S.S., Jones, W.D., van Kogelenberg, M., King, D.A., McRae, J., Morley, K.I., Parthiban, V., Al-Turki, S., Ambridge, K., et al. (2015). Large-scale discovery of novel genetic causes of developmental disorders. Nature 519, 223-228.). In our comparison, the Agilent v6 exome performs better than the NimbleGen's MedExome both in terms of efficiency and evenness of coverage distribution. With its larger target size, it is also more comprehensive, and therefore the better choice in research projects that aim to identify novel disease-associated genes. In contrast, if the exomes are mainly used in a diagnostic setting, we see advantages for the new NimbleGen MedExome. We find a superior coverage here in those genes of high clinical relevance that likely allows for a better detection of relevant, disease-causing mutations. [ABSTRACT FROM AUTHOR]

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules. [ABSTRACT FROM AUTHOR]

In 2014, a slew of structures of glutamate receptors were published, based on crystallography and electron microscopy. Here we review these insights, integrate them with existing knowledge about receptor function and try to understand how the structures relate to the key property of the AMPA receptor - its speed. [ABSTRACT FROM AUTHOR]

Fifteen years ago we discovered activity-dependent neuroprotective protein (ADNP), and showed that it is essential for brain formation/function. Our protein interaction studies identified ADNP as a member of the chromatin remodeling complex, SWI/SNF also associated with alternative splicing of tau and prediction of tauopathy. Recently, we have identified cytoplasmic ADNP interactions with the autophagy regulating microtubule-associated protein 1 light chain 3 (LC3) and with microtubule end-binding (EB) proteins. The ADNP-EB-binding SIP domain is shared with the ADNP snippet drug candidate, NAPVSIPQ termed NAP (davunetide). Thus, we identified a precise target for ADNP/NAP (davunetide) neuroprotection toward improved drug development. [ABSTRACT FROM AUTHOR]

Paul Ebensperger and Claudia Jessen-Trefzer take up another interesting aspect of artificial protein chemistry. In this context, Wang and coworkers describe a yeast three-hybrid system to identify the protein targets of small molecules, while addressing the challenge of compound uptake into yeast cells ([8]). This Highlight Issue of I Biological Chemistry i covers various aspects of today's drug discovery process at the interface between chemistry and biology, and provides an outlook on future research directions, methods and emerging trends. [Extracted from the article]

The article announces the decision by the publishers and editors of "Biological Chemistry" to publish accepted papers on its online content site. "Just Accepted" articles will now be published electronically in advance of their appearance in the journal's printed edition. The peer-reviewed "Just Accepted" articles will be accessible online soon after their final acceptance on the journal's online content site at http://www.atypon-link.com/WDG/loi/bchm.