Dynamics of endogenous Hsp70 synthesis in the brain of olfactory bulbectomized mice

Numerous epidemiological studies have established acute brain injury as one of the major risk factors for the Alzheimer''s disease (AD). However, the lack of animal models of AD-like degeneration triggered by a defined injury hampered the development of adequate therapies. Here we report that the surgical damage of the olfactory bulbs triggers the development of several pathologies, including amyloid-β accumulation and strong decrease of neuron density in the cortex and hippocampus as well as significant disturbance of spatial memory. Characteristically, these harmful consequences of the olfactory bulbectomy (OBX) have a peculiar dynamics in time with maximal manifestation in periods of 1–1.5 months and 8 months after the surgery and, hence, exhibit biphasic pattern with almost complete recovery period taking place at 5–6 months after the operation. The quantitative determination of endogenous inducible form of Hsp70 in different brain areas of OBX mice demonstrated characteristic fluctuations of Hsp70 levels depending on the time after the operation and age of mice. Interestingly, maximal induction of Hsp70 synthesis in the hippocampus exhibits clear-cut coincidence with the recovery period in OBX animals. The observed correlation enables to suggest curing effect of Hsp70 synthesis at an earlier period of pathology development and establishes it as a possible therapeutic agent for secondary grave consequences of brain injury, such as AD-like degeneration, for which neuroprotective therapy is urgently needed.     

Inhibition of Mycobacterium tuberculosis strains H37Rv and MDR MS-115 by a new set of C5 modified pyrimidine nucleosides

Two sets of pyrimidine nucleoside derivatives bearing extended alkyloxymethyl or alkyltriazolidomethyl substituents at position 5 of the nucleobase were synthesized and evaluated as potential antituberculosis agents. The impact of modifications at 3′- and 5′-positions of the carbohydrate moiety on the antimycobacterial activity and cytotoxicity was studied. The highest effect was shown for 5-dodecyloxymethyl-2′-deoxyuridine, 5-decyltriazolidomethyl-2′-deoxyuridine, and 5-dodecyltriazolidomethyl-2′-deoxycytidine. They effectively inhibited the growth of two Mycobacterium tuberculosis strains in vitro, laboratory H37Rv (MIC₉₉ = 20, 10, and 20 μg/mL, respectively) and clinical MDR MS-115 resistant to five top antituberculosis drugs (МIC₉₉ = 50, 10, and 10 μg/mL, respectively).     

Penetrating Cations Enhance Uncoupling Activity of Anionic Protonophores in Mitochondria

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H⁺ ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C₁₂TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C₁₂TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C₁₂TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.     

The SULFs,Extracellular Sulfatases for Heparan Sulfate,Promote the Migration of Corneal Epithelial Cells during Wound Repair

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1^−/−, but not Sulf2^−/−, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1^−/− mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.     

Characterization of chimpanzee/human monoclonal antibodies to vaccinia virus A33 glycoprotein and its variola virus homolog in vitro and in a vaccinia virus mouse protection model

Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma 1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (K(d) of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.     

Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.Despite excitement about the development of targeted therapy strategies for cancer, few cures have been achieved. In patients with BRAF mutant melanoma, treatment with small molecule BRAF inhibitors typically follows a course of response and tumor shrinkage followed by eventual relapse and resistance (mean progression-free survival is ∼5.3 months) (1). Resistance to BRAF inhibitors is typically accompanied by reactivation of the MAPK signaling pathway, an effect mediated through activating mutations in NRAS and MEK1/2, genomic amplification of BRAF, increased expression of CRAF and Cot, and the acquisition of BRAF splice-form mutants (2–5). There is also evidence that increased PI3K/AKT signaling, resulting from the genetic inactivation of PTEN and NF1 and increased receptor tyrosine kinase (RTK)¹ signaling, may be involved in acquired BRAF inhibitor resistance (5–7). Many of the signaling proteins implicated in the escape from BRAF inhibitor therapy are clients of heat shock protein (HSP)-90 (8). Preclinical evidence now indicates that HSP90 inhibitors can overcome acquired and intrinsic BRAF inhibitor resistance, and clinical trials have been initiated to evaluate the BRAF/HSP90 combination in newly diagnosed patients (8, 9).Although targeted therapy strategies have been promising in BRAF mutant melanoma, few options currently exist for the 15–20% of melanoma patients whose tumors harbor activating NRAS mutations (10). Although there is some evidence that MEK inhibitors have activity in NRAS mutant melanoma patients, responses tend to be short-lived (mean progression-free survival ∼3 months) and resistance is nearly inevitable (11). Our emerging experience suggests that oncogene-driven signaling networks are highly robust with the capacity to rapidly adapt (12, 13). The future success of targeted therapy for melanoma and other cancers will depend upon the development of strategies that identify and overcome these adaptive escape mechanisms.The evaluation of targeted therapy responses in patients has proved to be challenging. The clinical development of HSP90 inhibitors has been hampered in part by the lack of a good pharmacodynamic assay for measuring HSP90 inhibition within tumor specimens (14). Additionally, very little is known about the adaptive changes that occur following the inhibition of MEK/ERK signaling in NRAS mutant melanoma. To address these issues, the optimal technique is liquid chromatography-multiple reaction monitoring mass spectrometry, which been shown to be highly reproducible and portable across laboratories (15–18).In addition to these technical developments, LC-MRM has also been shown to have excellent application to the study of biological pathways, including phosphotyrosine signaling, β-catenin signaling in colon cancer, and the evasion of apoptosis following BRAF inhibition in PTEN null melanoma (19–21). This technique can also be readily translated from cell line models to patient specimens. Here, we have developed a novel multiplexed LC-MRM assay to quantify the expression of >80 key signaling proteins in cell line models and fine needle aspirates from accessible melanoma lesions (22). In this study, we present the proof-of-principle for monitoring multiple signaling proteins in melanomas treated with either HSP90 or MEK inhibitors. Through this method, we identify the degradation of key HSP90 client proteins in vivo and elucidate a novel mechanism of adaptation to MEK inhibition through increased RTK signaling.     

Survival of mycobacteria depends on proteasome‐mediated amino acid recycling under nutrient limitation

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S‐proteasome, bacterial prokaryotic ubiquitin‐like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup‐proteasome system (PPS) is important for virulence, yet its physiological role in non‐pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto‐regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.     

RNA Binding and Core Complexes Constitute the U-Insertion/Deletion Editosome

Enzymes embedded into the RNA editing core complex (RECC) catalyze the U-insertion/deletion editing cascade to generate open reading frames in trypanosomal mitochondrial mRNAs. The sequential reactions of mRNA cleavage, U-addition or removal, and ligation are directed by guide RNAs (gRNAs). We combined proteomic, genetic, and functional studies with sequencing of total and complex-bound RNAs to define a protein particle responsible for the recognition of gRNAs and pre-mRNA substrates, editing intermediates, and products. This approximately 23-polypeptide tripartite assembly, termed the RNA editing substrate binding complex (RESC), also functions as the interface between mRNA editing, polyadenylation, and translation. Furthermore, we found that gRNAs represent only a subset of small mitochondrial RNAs, and yet an inexplicably high fraction of them possess 3′ U-tails, which correlates with gRNA''s enrichment in the RESC. Although both gRNAs and mRNAs are associated with the RESC, their metabolic fates are distinct: gRNAs are degraded in an editing-dependent process, whereas edited mRNAs undergo 3′ adenylation/uridylation prior to translation. Our results demonstrate that the well-characterized editing core complex (RECC) and the RNA binding particle defined in this study (RESC) typify enzymatic and substrate binding macromolecular constituents, respectively, of the ∼40S RNA editing holoenzyme, the editosome.