Humanization of N- and O-glycosylated plant proteins

Glikanske strukture terapeutskih proteina utječu na njihova farmakodinamička i farmakokinetička svojstva. Optimalna glikozilacija povećala bi učinkovitost i sigurnost rekombinantnih farmaceutskih proteina. S obzirom na jeftiniji uzgoj i procesiranje, jednostavan N-glikozilacijski put i odsutnost O-glikozilacijskog puta karakterističnog za sisavce, biljke su se posljednjih godina nametnule u modeliranju ekspresijskog sustava za proizvodnju potpuno humaniziranih rekombinantnih terapeutskih proteina. Ciljevi pri konstruiranju takvog optimalnog sustava su inaktivacija gena za enzime karakteristične za biljnu glikozilaciju te uvođenje gena za enzime koji su karakteristični za čovjeka.Glycan structures of therapeutic proteins affect their pharmacodynamics and pharmacokinetic characteristics. Optimal glycosylation would enhance efficiency and safety of recombinant pharmaceutical proteins. Due to inexpensive and simple cultivation, simple Nglycosylation pathway and absence of typical mammalian O-glycosylation pathway, in recent years plants have emerged as interesting new hosts for the production of fully humanized recombinant pharmaceutical proteins. Aims in engineering optimal expression system are inactivation of genes for plant-specific enzymes and introduction of enzymes that are humanspecific

PI3K/Akt and ERK1/2 Signalling Are Involved in Quercetin-Mediated Neuroprotection against Copper-Induced Injury

Copper, a transition metal with essential cellular functions, exerts neurotoxic effects when present in excess by promoting production of reactive oxygen species (ROS). The aim of the present study was to investigate potential benefits of flavonoid quercetin against copper-induced toxicity. Results obtained with MTT assay indicate that the effects of quercetin are determined by the severity of the toxic insult. In moderately injured P19 neuronal cells, concomitant treatment with 150 μM quercetin improved viability by preventing ROS formation, caspase-3 activation, and chromatin condensation. Western blot analysis revealed that quercetin reduced copper-induced increase in p53 upregulated modulator of apoptosis (PUMA) expression and promoted upregulation of nucleoside diphosphate kinase NME1. Levels of p53 and Bax proteins were not affected by both copper and quercetin. UO126 and wortmannin, inhibitors of ERK1/2 and PI3K/Akt signalling pathways, respectively, prevented neuroprotective effects of quercetin. In severely injured neurons, 30 μM quercetin exerted strong prooxidative action and exacerbated cytotoxic effects of copper, whereas 150 μM quercetin failed to affect neuronal survival. These results demonstrate the dual nature of quercetin action in copper-related neurodegeneration. Hence, they are relevant in the context of considering quercetin as a possible therapeutic for neuroprotection and imply that detailed pharmacological and toxicological studies must be carried out for natural compounds capable of acting both as antioxidants and prooxidants

Effect of quercetin on oxidative stress in P19 neurons in the presence of copper ions

Mnoga istraživanja pokazala su povezanost promijenjene regulacije homeostaze bakra s nastankom i progresijom neurodegenerativnih bolesti. Toksična svojstva bakra uglavnom se pripisuju povećanoj proizvodnji reaktivnih kisikovih vrsta i posljedičnom oksidacijskom stresu. Cilj istraživanja bio je analizirati učinak biljnog flavonoida kvercetina na neurone P19 u kojima je stanje oksidacijskog stresa izazvano suviškom iona bakra. U najvećoj primijenjenoj koncentraciji (150 μM) kvercetin je povećao preživljenje neurona P19 tretiranih s 0,5 mM CuSO4 te smanjio bakrom izazvan porast nakupljanja reaktivnih kisikovih vrsta. Analiza metodom Western blotting pokazala je da suvišak bakra dovodi do porasta ekspresije proapoptotskog proteina PUMA, te da je u prisutnosti kvercetina taj porast smanjen. Ovakvi rezultatu upućuje na antiapoptotski mehanizam djelovanja kvercetina. U neuronima P19 kvercetin je također inducirao ekspresiju nukleozid difosfat kinaze NME1. Promjene u ekspresiji proteina p53 i Bax nisu nađene. Dobiveni rezultati pokazuju neuroprotektivni učinak kvercetina u uvjetima oksidacijskog stresa izazvanog ionima bakra i upućuju na antioksidacijski i antiapoptotski mehanizam njegova djelovanja.A growing number of studies suggest that the deregulation of copper homeostasis is an important factor in the onset and progression of neurodegenerative diseases. Toxic effects of copper are mainly attributed to the increased production of reactive oxygen species (ROS) and subsequent oxidative stress. The aim of this study was to analyse the effect of the plant-derived flavonoid quercetin on P19 neurons, in which the oxidative stress was induced by exposure to the excess copper ions. In the highest applied concentration (150 μM) of quercetin the viability of P19 neurons treated with 0.5 mM CuSO4 was increased and the copper-induced accumulation of reactive oxygen species was prevented. Western blotting analysis showed that copper increases expression of proapoptotic protein PUMA, whereas in the presence of quercetin the obtained increase was diminished. These results indicate anti-apoptotic mechanism of quercetin action. In P19 neurons quercetin also induced the expression of nucleoside diphosphate kinase NME1. The expression of p53 and Bax remained unchanged at the protein level. The obtained results demonstrate neuroprotective effect of quercetin against oxidative stress induced by exposure to copper ions, and suggest antioxidative and antiapoptotic mechanism of its action

Effect of quercetin on oxidative stress in P19 neurons in the presence of copper ions

Mnoga istraživanja pokazala su povezanost promijenjene regulacije homeostaze bakra s nastankom i progresijom neurodegenerativnih bolesti. Toksična svojstva bakra uglavnom se pripisuju povećanoj proizvodnji reaktivnih kisikovih vrsta i posljedičnom oksidacijskom stresu. Cilj istraživanja bio je analizirati učinak biljnog flavonoida kvercetina na neurone P19 u kojima je stanje oksidacijskog stresa izazvano suviškom iona bakra. U najvećoj primijenjenoj koncentraciji (150 μM) kvercetin je povećao preživljenje neurona P19 tretiranih s 0,5 mM CuSO4 te smanjio bakrom izazvan porast nakupljanja reaktivnih kisikovih vrsta. Analiza metodom Western blotting pokazala je da suvišak bakra dovodi do porasta ekspresije proapoptotskog proteina PUMA, te da je u prisutnosti kvercetina taj porast smanjen. Ovakvi rezultatu upućuje na antiapoptotski mehanizam djelovanja kvercetina. U neuronima P19 kvercetin je također inducirao ekspresiju nukleozid difosfat kinaze NME1. Promjene u ekspresiji proteina p53 i Bax nisu nađene. Dobiveni rezultati pokazuju neuroprotektivni učinak kvercetina u uvjetima oksidacijskog stresa izazvanog ionima bakra i upućuju na antioksidacijski i antiapoptotski mehanizam njegova djelovanja.A growing number of studies suggest that the deregulation of copper homeostasis is an important factor in the onset and progression of neurodegenerative diseases. Toxic effects of copper are mainly attributed to the increased production of reactive oxygen species (ROS) and subsequent oxidative stress. The aim of this study was to analyse the effect of the plant-derived flavonoid quercetin on P19 neurons, in which the oxidative stress was induced by exposure to the excess copper ions. In the highest applied concentration (150 μM) of quercetin the viability of P19 neurons treated with 0.5 mM CuSO4 was increased and the copper-induced accumulation of reactive oxygen species was prevented. Western blotting analysis showed that copper increases expression of proapoptotic protein PUMA, whereas in the presence of quercetin the obtained increase was diminished. These results indicate anti-apoptotic mechanism of quercetin action. In P19 neurons quercetin also induced the expression of nucleoside diphosphate kinase NME1. The expression of p53 and Bax remained unchanged at the protein level. The obtained results demonstrate neuroprotective effect of quercetin against oxidative stress induced by exposure to copper ions, and suggest antioxidative and antiapoptotic mechanism of its action

Effect of quercetin on oxidative stress in P19 neurons in the presence of copper ions

Mnoga istraživanja pokazala su povezanost promijenjene regulacije homeostaze bakra s nastankom i progresijom neurodegenerativnih bolesti. Toksična svojstva bakra uglavnom se pripisuju povećanoj proizvodnji reaktivnih kisikovih vrsta i posljedičnom oksidacijskom stresu. Cilj istraživanja bio je analizirati učinak biljnog flavonoida kvercetina na neurone P19 u kojima je stanje oksidacijskog stresa izazvano suviškom iona bakra. U najvećoj primijenjenoj koncentraciji (150 μM) kvercetin je povećao preživljenje neurona P19 tretiranih s 0,5 mM CuSO4 te smanjio bakrom izazvan porast nakupljanja reaktivnih kisikovih vrsta. Analiza metodom Western blotting pokazala je da suvišak bakra dovodi do porasta ekspresije proapoptotskog proteina PUMA, te da je u prisutnosti kvercetina taj porast smanjen. Ovakvi rezultatu upućuje na antiapoptotski mehanizam djelovanja kvercetina. U neuronima P19 kvercetin je također inducirao ekspresiju nukleozid difosfat kinaze NME1. Promjene u ekspresiji proteina p53 i Bax nisu nađene. Dobiveni rezultati pokazuju neuroprotektivni učinak kvercetina u uvjetima oksidacijskog stresa izazvanog ionima bakra i upućuju na antioksidacijski i antiapoptotski mehanizam njegova djelovanja.A growing number of studies suggest that the deregulation of copper homeostasis is an important factor in the onset and progression of neurodegenerative diseases. Toxic effects of copper are mainly attributed to the increased production of reactive oxygen species (ROS) and subsequent oxidative stress. The aim of this study was to analyse the effect of the plant-derived flavonoid quercetin on P19 neurons, in which the oxidative stress was induced by exposure to the excess copper ions. In the highest applied concentration (150 μM) of quercetin the viability of P19 neurons treated with 0.5 mM CuSO4 was increased and the copper-induced accumulation of reactive oxygen species was prevented. Western blotting analysis showed that copper increases expression of proapoptotic protein PUMA, whereas in the presence of quercetin the obtained increase was diminished. These results indicate anti-apoptotic mechanism of quercetin action. In P19 neurons quercetin also induced the expression of nucleoside diphosphate kinase NME1. The expression of p53 and Bax remained unchanged at the protein level. The obtained results demonstrate neuroprotective effect of quercetin against oxidative stress induced by exposure to copper ions, and suggest antioxidative and antiapoptotic mechanism of its action

Nuclear Ubiquitin-Proteasome Pathways in Proteostasis Maintenance

Protein homeostasis, or proteostasis, is crucial for the functioning of a cell, as proteins that are mislocalized, present in excessive amounts, or aberrant due to misfolding or other type of damage can be harmful. Proteostasis includes attaining the correct protein structure, localization, and the formation of higher order complexes, and well as the appropriate protein concentrations. Consequences of proteostasis imbalance are evident in a range of neurodegenerative diseases characterized by protein misfolding and aggregation, such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis. To protect the cell from the accumulation of aberrant proteins, a network of protein quality control (PQC) pathways identifies the substrates and direct them towards refolding or elimination via regulated protein degradation. The main pathway for degradation of misfolded proteins is the ubiquitin-proteasome system. PQC pathways have been first described in the cytoplasm and the endoplasmic reticulum, however, accumulating evidence indicates that the nucleus is an important PQC compartment for ubiquitination and proteasomal degradation of not only nuclear, but also cytoplasmic proteins. In this review, we summarize the nuclear ubiquitin-proteasome pathways involved in proteostasis maintenance in yeast, focusing on inner nuclear membrane-associated degradation (INMAD) and San1-mediated protein quality control

Metals in Alzheimer’s Disease

The role of metals in the pathogenesis of Alzheimer’s disease (AD) is still debated. Although previous research has linked changes in essential metal homeostasis and exposure to environmental heavy metals to the pathogenesis of AD, more research is needed to determine the relationship between metals and AD. In this review, we included human studies that (1) compared the metal concentrations between AD patients and healthy controls, (2) correlated concentrations of AD cerebrospinal fluid (CSF) biomarkers with metal concentrations, and (3) used Mendelian randomization (MR) to assess the potential metal contributions to AD risk. Although many studies have examined various metals in dementia patients, understanding the dynamics of metals in these patients remains difficult due to considerable inconsistencies among the results of individual studies. The most consistent findings were for Zn and Cu, with most studies observing a decrease in Zn levels and an increase in Cu levels in AD patients. However, several studies found no such relation. Because few studies have compared metal levels with biomarker levels in the CSF of AD patients, more research of this type is required. Given that MR is revolutionizing epidemiologic research, additional MR studies that include participants from diverse ethnic backgrounds to assess the causal relationship between metals and AD risk are critical

Anterograde and Retrograde Propagation of Inoculated Human Tau Fibrils and Tau Oligomers in a Non-Transgenic Rat Tauopathy Model

The tauopathy of Alzheimer’s disease (AD) is first observed in the brainstem and entorhinal cortex, spreading trans-synaptically along specific pathways to other brain regions with recognizable patterns. Tau propagation occurs retrogradely and anterogradely (trans-synaptically) along a given pathway and through exosomes and microglial cells. Some aspects of in vivo tau spreading have been replicated in transgenic mice models expressing a mutated human MAPT (tau) gene and in wild-type mice. In this study, we aimed to characterize the propagation of different forms of tau species in non-transgenic 3–4 months old wild-type rats after a single unilateral injection of human tau oligomers and tau fibrils into the medial entorhinal cortex (mEC). We determined whether different variants of the inoculated human tau protein, tau fibrils, and tau oligomers, would induce similar neurofibrillary changes and propagate in an AD-related pattern, and how tau-related pathological changes would correlate with presumed cognitive impairment. We injected human tau fibrils and tau oligomers stereotaxically into the mEC and examined the distribution of tau-related changes at 3 days and 4, 8, and 11 months post-injection using antibodies AT8 and MC1, which reveal early phosphorylation and aberrant conformation of tau, respectively, HT7, anti-synaptophysin, and the Gallyas silver staining method. Human tau oligomers and tau fibrils exhibited some similarities and some differences in their ability to seed and propagate tau-related changes. Both human tau fibrils and tau oligomers rapidly propagated from the mEC anterogradely into the hippocampus and various parts of the neocortex. However, using a human tau-specific HT7 antibody, 3 days post-injection we found inoculated human tau oligomers in the red nucleus, primary motor, and primary somatosensory cortex, a finding not seen in animals inoculated with human tau fibrils. In animals inoculated with human tau fibrils, 3 days post-injection the HT7 antibody showed fibrils in the pontine reticular nucleus, a finding explained only by uptake of human tau fibrils by incoming presynaptic fibers to the mEC and retrograde transport of inoculated human tau fibrils to the brainstem. Rats inoculated with human tau fibrils showed as early as 4 months after inoculation a spread of phosphorylated tau protein at the AT8 epitopes throughout the brain, dramatically faster propagation of neurofibrillary changes than with human tau oligomers. The overall severity of tau protein changes 4, 8, and 11 months after inoculation of human tau oligomers and tau fibrils correlated well with spatial working memory and cognition impairments, as measured by the T-maze spontaneous alternation, novel object recognition, and object location tests. We concluded that this non-trangenic rat model of tauopathy, especially when using human tau fibrils, demonstrates rapidly developing pathologic alterations in neurons, synapses, and identifiable pathways together with cognitive and behavioral changes, through the anterograde and retrograde spreading of neurofibrillary degeneration. Therefore, it represents a promising model for future experimental studies of primary and secondary tauopathies, especially AD