| 1. |
- Davies, Victoria S., et al.
(author)
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Repeated short excursions from thermoneutrality suffice to restructure brown adipose tissue
- 2023
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In: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 210, s. 40-49
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Journal article (peer-reviewed)abstract
- Given the presence of brown adipose tissue in adult humans, an important issue is whether human brown adipose tissue is recruitable. Cold exposure is the canonical recruitment treatment; however, in experimental animals (mice), recruitment of brown adipose tissue is normally induced by placing the mice in constant cold, a procedure not feasible in humans. For possible translational applications, we have therefore investigated whether shorter daily excursions from thermoneutrality would suffice to qualitatively and quantitatively induce recruitment in mice. Mice, housed at thermoneutrality (30 °C) to mimic human conditions, were transferred every day for 4 weeks to cool conditions (18 °C), for 0, 15, 30, 120 and 420 min (or placed constantly in 18 °C). On the examination day, the mice were not exposed to cold. Very short daily exposures (≤30 minutes) were sufficient to induce structural changes in the form of higher protein density in brown adipose tissue, changes that may affect the identification of the tissue in e.g. computer tomography and other scan studies. To estimate thermogenic capacity, UCP1 protein levels were followed. No UCP1 protein was detectable in inguinal white adipose tissue. In the interscapular brown adipose tissue, a remarkable two-phase reaction was seen. Very short daily exposures (≤30 minutes) were sufficient to induce a significant increase in total UCP1 levels. For attainment of full cold acclimation, the mice had, however, to remain exposed to the cold. The studies indicate that marked alterations in brown adipose tissue composition can be induced in mammals through relatively modest stimulation events.
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| 2. |
- Dhumal, Tushar Tukaram, et al.
(author)
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Molecular explorations of the Leishmania donovani 6-phosphogluconolactonase enzyme, a key player in the pentose phosphate pathway
- 2022
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In: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 202, s. 212-225
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Journal article (peer-reviewed)abstract
- The enzymes of the pentose phosphate pathway are vital to survival in kinetoplastids. The second step of the pentose phosphate pathway involves hydrolytic cleavage of 6-phosphogluconolactone to 6- phosphogluconic acid by 6-phosphogluconolactonase (6PGL). In the present study, Leishmania dono-vani 6PGL (Ld6PGL) was cloned and overexpressed in bacterial expression system. Comparative sequence analysis revealed the conserved sequence motifs, functionally and structurally important residues in 6PGL family. In silico amino acid substitution study and interacting partners of 6PGL were predicted. The Ld6PGL enzyme was found to be active in the assay and in the parasites. Specificity was confirmed by Western blot analysis. The similar to 30 kDa protein was found to be a dimer in MALDI, glutaraldehyde cross-linking and size exclusion chromatography studies. Kinetic analysis and structural stability studies of Ld6PGL were performed with denaturants and at varied temperature. Computational 3D Structural modelling of Ld6PGL elucidates that it has a similar a/b hydrolase fold structural topology as in other members of 6PGL family. The three loops are found in extended form when the structure is compared with the human 6PGL (Hs6PGL). Further, enzyme substrate binding mode and its mechanism were investigated using the molecular docking and molecular simulation studies. Interesting dynamics action of substrate 6-phosphogluconolactone was observed into active site during MD simulation. Interesting differences were observed between host and parasite enzyme which pointed towards its potential to be explored as an antileishmanial drug target. This study forms the basis for further analysis of the role of Ld6PGL in combating oxidative stress in Leishmania.
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| 3. |
- Jamroskovic, Jan, et al.
(author)
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Probing the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution
- 2022
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In: Biochimie. - : Elsevier. - 0300-9084 .- 1638-6183. ; 199, s. 81-91
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Journal article (peer-reviewed)abstract
- Cytosine-rich DNA can fold into four-stranded intercalated structures called i-motifs (iMs) under acidic conditions through the formation of hemi-protonated C:C+ base pairs. However, the folding and stability of iMs rely on many other factors that are not yet fully understood. Here, we combined biochemical and biophysical approaches to determine the factors influencing iM stability under a wide range of experimental conditions. By using high-resolution primer extension assays, circular dichroism, and absorption spectroscopies, we demonstrate that the stabilities of three different biologically relevant iMs are not dependent on molecular crowding agents. Instead, some of the crowding agents affected overall DNA synthesis. We also tested a range of small molecules to determine their effect on iM stabilization at physiological temperature and demonstrated that the G-quadruplex-specific molecule CX-5461 is also a promising candidate for selective iM stabilization. This work provides important insights into the requirements needed for different assays to accurately study iM stabilization, which will serve as important tools for understanding the contribution of iMs in cell regulation and their potential as therapeutic targets.
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| 4. |
- Lagunas-Rangel, Francisco Alejandro
(author)
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KDM6B (JMJD3) and its dual role in cancer
- 2021
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In: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 184, s. 63-71
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Journal article (peer-reviewed)abstract
- Epigenetic modifications play a fundamental role in the regulation of gene expression and cell fate. During the development of cancer, epigenetic modifications appear that favor cell proliferation and migration, but at the same time prevent differentiation and apoptosis, among other processes. KDM6B is a histone demethylase that specifically removes methyl groups from H3K27me3, thus allowing re-expression of its target genes. It is currently known that KDM6B can act as both a tumor suppressor and an oncogene depending on the cellular context. Therefore, in this work we summarize the current knowledge of the role that KDM6B plays in different oncological contexts, and we try to orient it towards its clinical application.
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| 5. |
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| 6. |
- Li, He, D.Sc, 1985-, et al.
(author)
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Family 92 carbohydrate-binding modules specific for β-1,6-glucans increase the thermostability of a bacterial chitinase
- 2023
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In: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 212, s. 153-160
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Journal article (peer-reviewed)abstract
- In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10–15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.
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| 7. |
- Maltseva, Diana, et al.
(author)
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Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy
- 2020
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In: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 174, s. 107-116
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Journal article (peer-reviewed)abstract
- The interaction of tumor cells with the extracellular matrix (ECM) may affect the rate of cancer progression and metastasis. One of the major components of ECM are laminins, the heterotrimeric glycoproteins consisting of α-, β-, and γ-chains (αβγ). Laminins interact with their cell surface receptors and, thus, regulate multiple cellular processes. In this work, we demonstrate that shRNA-mediated knockdown of the α5 laminin chain results in Wnt- and mTORC1-dependent partial dedifferentiation of colorectal cancer cells. Furthermore, we showed that this dedifferentiation involved activation of ER-stress signaling, pathway promoting the sensitivity of cells to 5-fluorouracil.
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| 8. |
- Pata, Jorgaq, et al.
(author)
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Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species
- 2024
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In: Biochimie. - 0300-9084 .- 1638-6183. ; 220, s. 167-178
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Journal article (peer-reviewed)abstract
- Candida albicans and C. glabrata express exporters of the ATP -binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C -terminal end with either a His -tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal -affinity and size -exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.
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| 9. |
- Rodin, Sergey, et al.
(author)
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Aberrant interactions between amyloid-beta and alpha5 laminins as possible driver of neuronal disfunction in Alzheimer's disease
- 2020
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In: Biochimie. - : Elsevier. - 0300-9084 .- 1638-6183. ; 174, s. 44-48
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Research review (peer-reviewed)abstract
- It has been widely accepted that laminins are involved in pathogenesis of Alzheimer's disease (AD). Amyloid plaques in AD patients are associated with immunostaining using antibodies raised against laminin-111, and laminin-111 has been shown to prevent aggregation of amyloid peptides. Although numerous articles describe small peptides from laminin-111 that are capable to disaggregate amyloid buildups and reduce neurotoxicity in in vitro and in vivo models, there is no approved laminin-111-based therapeutic approaches for treatment of AD. Also, it has been shown that immunoreactivity to laminin111 appears late in development of cerebral amyloidosis. Based on the published data, we hypothesize that aberrant interaction between amyloid-beta and alpha 5-laminins such as laminin-511 prevents the necessary laminin signaling into neurons leading to neurodegeneration and contributing to the early development of AD. Laminin-511 is the key extracellular protein that protects neurons from anoikis, inhibits excitoxicity and provides signaling that stabilizes dendritic spines and synapses in the developed brain. Absence of the signaling from laminin-511 leads to behavioral defects in mice. Laminin-511 and hippocampal neurons are in direct contact and accumulation of amyloid-beta that has been shown to avidly bind laminin-511 may physically decouple the interaction between alpha 5-laminins and the neuronal membrane receptors inhibiting the signaling. Under this hypothesis, protein domains and peptides from laminin alpha 5 chain may have a therapeutic potential in treatment of AD and the appearance of laminin-111 in the amyloid plaques is simply a consequence of the disease.
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| 10. |
- Verma, Swati, et al.
(author)
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Current perspectives for microbial lipases from extremophiles and metagenomics
- 2021
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In: Biochimie. - : Elsevier. - 0300-9084 .- 1638-6183. ; 182, s. 23-36
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Research review (peer-reviewed)abstract
- Microbial lipases are most broadly used biocatalysts for environmental and industrial applications. Lipases catalyze the hydrolysis and synthesis of long acyl chain esters and have a characteristic folding pattern of α/β hydrolase with highly conserved catalytic triad (Serine, Aspartic/Glutamic acid and Histidine). Mesophilic lipases (optimal activity in neutral pH range, mesophilic temperature range, atmospheric pressure, normal salinity, non-radio-resistant, and instability in organic solvents) have been in use for many industrial biotransformation reactions. However, lipases from extremophiles can be used to design biotransformation reactions with higher yields, less byproducts or useful side products and have been predicted to catalyze those reactions also, which otherwise are not possible with the mesophilic lipases. The extremophile lipase perform activity at extremes of temperature, pH, salinity, and pressure which can be screened from metagenome and de novo lipase design using computational approaches. Despite structural similarity, they exhibit great diversity at the sequence level. This diversity is broader when lipases from the bacterial, archaeal, plant, and animal domains/kingdoms are compared. Furthermore, a great diversity of novel lipases exists and can be discovered from the analysis of the dark matter - the unexplored nucleotide/metagenomic databases. This review is an update on extremophilic microbial lipases, their diversity, structure, and classification. An overview on novel lipases which have been detected through analysis of the genomic dark matter (metagenome) has also been presented.
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