Purple photosynthetic bacteria (PPB) are versatile microorganisms capable of producing various value-added chemicals, e.g., biopolymers and biofuels. They employ diverse metabolic pathways, allowing them to adapt to various growth conditions and even extreme environments. Thus, they are ideal organisms for the Next Generation Industrial Biotechnology concept of reducing the risk of contamination by using naturally robust extremophiles. Unfortunately, the potential of PPB for use in biotechnology is hampered by missing knowledge on regulations of their metabolism. Although Rhodospirillum rubrum represents a model purple bacterium studied for polyhydroxyalkanoate and hydrogen production, light/chemical energy conversion, and nitrogen fixation, little is known regarding the regulation of its metabolism at the transcriptomic level. Using RNA sequencing, we compared gene expression during the cultivation utilizing fructose and acetate as substrates in case of the wild-type strain R. rubrum DSM 467T and its knock-out mutant strain that is missing two polyhydroxyalkanoate synthases PhaC1 and PhaC2. During this first genome-wide expression study of R. rubrum, we were able to characterize cultivation-driven transcriptomic changes and to annotate non-coding elements as small RNAs.
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Potassium is an essential intracellular ion, and a sufficient intracellular concentration of it is crucial for many processes; therefore it is fundamental for cells to precisely regulate K+ uptake and efflux through the plasma membrane. The uniporter Trk1 is a key player in K+ acquisition in yeasts. The TRK1 gene is expressed at a low and stable level; thus the activity of the transporter needs to be regulated at a posttranslational level. S. cerevisiae Trk1 changes its activity and affinity for potassium ion quickly and according to both internal and external concentrations of K+, as well as the membrane potential. The molecular basis of these changes has not been elucidated, though phosphorylation is thought to play an important role. In this study, we examined the role of the second, short, and highly conserved intracellular hydrophilic loop of Trk1 (IL2), and identified two phosphorylable residues (Ser882 and Thr900) as very important for 1) the structure of the loop and consequently for the targeting of Trk1 to the plasma membrane, and 2) the upregulation of the transporter's activity reaching maximal affinity under low external K+ conditions. Moreover, we identified three residues (Thr155, Ser414, and Thr900) within the Trk1 protein as strong candidates for interaction with 14-3-3 regulatory proteins, and showed, in an in vitro experiment, that phosphorylated Thr900 of the IL2 indeed binds to both isoforms of yeast 14-3-3 proteins, Bmh1 and Bmh2.
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High-Risk neuroblastoma (NB) survival rate is still <50%, despite treatments being more and more aggressive. The biggest hurdle liable to cancer therapy failure is the drug resistance by tumor cells that is likely due to the intra-tumor heterogeneity (ITH). To investigate the link between ITH and therapy resistance in NB, we performed a single cell RNA sequencing (scRNAseq) of etoposide and cisplatin resistant NB and their parental cells. Our analysis showed a clear separation of resistant and parental cells for both conditions by identifying 8 distinct tumor clusters in etoposide-resistant/parental and 7 in cisplatin-resistant/parental cells. We discovered that drug resistance can affect NB cell identities; highlighting the bi-directional ability of adrenergic-to-mesenchymal transition of NB cells. The biological processes driving the identified resistant cell subpopulations revealed genes such as (BARD1, BRCA1, PARP1, HISTH1 axis, members of RPL family), suggesting a potential drug resistance due to the acquisition of DNA repair mechanisms and to the modification of the drug targets. Deconvolution analysis of bulk RNAseq data from 498 tumors with cell subpopulation signatures showed that the transcriptional heterogeneity of our cellular models reflected the ITH of NB tumors and allowed the identification of clusters associated with worse/better survival. Our study demonstrates the distinct cell populations characterized by genes involved in different biological processes can have a role in NB drug treatment failure. These findings evidence the importance of ITH in NB drug resistance studies and the chance that scRNA-seq analysis offers in the identification of genes and pathways liable for drug resistance.
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Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over non-specific factors because of lower rates of deleterious side effects. Specifically, staphylokinase (SAK) is a pharmacologically attractive indirect plasminogen activator protein of bacterial origin that forms stoichiometric noncovalent complexes with plasmin, promoting the conversion of plasminogen into plasmin. Here we report a computer-assisted re-design of the molecular surface of SAK to increase its affinity for plasmin. A set of computationally designed SAK mutants was produced recombinantly and biochemically characterized. Screening revealed a pharmacologically interesting SAK mutant with ∼7-fold enhanced affinity toward plasmin, ∼10-fold improved plasmin selectivity and moderately higher plasmin-generating efficiency in vitro. Collectively, the results obtained provide a framework for SAK engineering using computational affinity-design that could pave the way to next-generation of effective, highly selective, and less toxic thrombolytics.
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In a single human gut, which is estimated to produce 1000-times more bacteria in a single day than the entire human population on Earth as of 2020, the potential for evolution is vast. In addition to the sheer volume of reproductive events, prokaryotes can transfer most genes horizontally, greatly accelerating their potential to evolve. In the face of this evolutionary potential, Westernization has led to profound changes in the ecosystem of the gut, including increased chronic inflammation in many individuals and dramatically reduced fiber consumption and decreased seasonal variation in the diet of most individuals. Experimental work using a variety of model systems has shown that bacteria will evolve within days to weeks when faced with substantial environmental changes. However, studies evaluating the effects of inflammation of the gut on the microbiota are still in their infancy and generally confounded by the effects of the microbiota on the immune system. At the same time, experimental data indicate that complete loss of fiber from the diet constitutes an extinction-level event for the gut microbiota. However, these studies evaluating diet may not apply to Westernized humans who typically have reduced but not absent levels of fiber in their diet. Thus, while it is expected that the microbiota will evolve rapidly in the face of Westernization, experimental studies that address the magnitude of that evolution are generally lacking, and it remains unknown to what extent this evolutionary process affects disease and the ability to treat the disease state.
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Single-celled yeasts form spatially structured populations - colonies and biofilms, either alone (single-species biofilms) or in cooperation with other microorganisms (mixed-species biofilms). Within populations, yeast cells develop in a coordinated manner, interact with each other and differentiate into specialized cell subpopulations that can better adapt to changing conditions (e.g. by reprogramming metabolism during nutrient deficiency) or protect the overall population from external influences (e.g. via extracellular matrix). Various omics tools together with specialized techniques for separating differentiated cells and in situ microscopy have revealed important processes and cell interactions in these structures, which are summarized here. Nevertheless, current knowledge is still only a small part of the mosaic of complexity and diversity of the multicellular structures that yeasts form in different environments. Future challenges include the use of integrated multi-omics approaches and a greater emphasis on the analysis of differentiated cell subpopulations with specific functions.
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Cancer cells can escape the effects of chemotherapy through mutations and upregulation of a tyrosine kinase protein called the epidermal growth factor receptor (EGFR). In the past two decades, four generations of tyrosine kinase inhibitors targeting EGFR have been developed. Using comparative structure analysis of 116 EGFR-drug complex crystal structures, cluster analysis produces two clans of 73 and 43 structures, respectively. The first clan of 73 structures is larger and is comprised mostly of the C-helix-IN conformation while the second clan of 43 structures correlates with the C-helix-OUT conformation. A deep rotamer analysis identifies 43 residues (18%) of the total of 237 residues spanning the kinase structures under investigation with significant rotamer variations between the C-helix-IN and C-helix-OUT clans. The locations of these rotamer variations take on the appearance of side chain conformational relays extending out from points of EGFR mutation to different regions of the EGFR kinase. Accordingly, we propose that key EGFR mutations act singly or together to induce drug resistant conformational changes in EGFR that are communicated via these side chain conformational relays. Accordingly, these side chain conformational relays appear to play a significant role in the development of tumour resistance. This phenomenon also suggests a new paradigm in protein conformational change that is mediated by supportive relays of rotamers on the protein surface, rather than through conventional backbone movements.
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The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8-10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.
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The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pathological pulmonary symptoms. Most efforts to develop vaccines and drugs against this virus target the spike glycoprotein, particularly its S1 subunit, which is recognised by angiotensin-converting enzyme 2. Here we use the in-house developed tool CaverDock to perform virtual screening against spike glycoprotein using a cryogenic electron microscopy structure (PDB-ID: 6VXX) and the representative structures of five most populated clusters from a previously published molecular dynamics simulation. The dataset of ligands was obtained from the ZINC database and consists of drugs approved for clinical use worldwide. Trajectories for the passage of individual drugs through the tunnel of the spike glycoprotein homotrimer, their binding energies within the tunnel, and the duration of their contacts with the trimer's three subunits were computed for the full dataset. Multivariate statistical methods were then used to establish structure-activity relationships and select top candidate for movement inhibition. This new protocol for the rapid screening of globally approved drugs (4359 ligands) in a multi-state protein structure (6 states) showed high robustness in the rate of finished calculations. The protocol is universal and can be applied to any target protein with an experimental tertiary structure containing protein tunnels or channels. The protocol will be implemented in the next version of CaverWeb (https://loschmidt.chemi.muni.cz/caverweb/) to make it accessible to the wider scientific community.
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Repeat-rich regions of higher plant genomes are usually associated with constitutive heterochromatin, a specific type of chromatin that forms tightly packed nuclear chromocenters and chromosome bands. There is a large body of cytogenetic evidence that these chromosome regions are often composed of tandemly organized satellite DNA. However, comparatively little is known about the sequence arrangement within heterochromatic regions, which are difficult to assemble due to their repeated nature. Here, we explore long-range sequence organization of heterochromatin regions containing the major satellite repeat CUS-TR24 in the holocentric plant Cuscuta europaea. Using a combination of ultra-long read sequencing with assembly-free sequence analysis, we reveal the complex structure of these loci, which are composed of short arrays of CUS-TR24 interrupted frequently by emerging simple sequence repeats and targeted insertions of a specific lineage of LINE retrotransposons. These data suggest that the organization of satellite repeats constituting heterochromatic chromosome bands can be more complex than previously envisioned, and demonstrate that heterochromatin organization can be efficiently investigated without the need for genome assembly.
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