@article{ZhangHubalewskaIgnatova2009, author = {Zhang, Gong and Hubalewska, Magdalena and Ignatova, Zoya}, title = {Transient ribosomal attenuation coordinates protein synthesis and co-translational folding}, issn = {1545-9985}, doi = {10.1038/Nsmb.1554}, year = {2009}, abstract = {Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein Sufl can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.}, language = {en} } @article{CzechWendeMoerletal.2013, author = {Czech, Andreas and Wende, Sandra and Moerl, Mario and Pan, Tao and Ignatova, Zoya}, title = {Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress}, series = {PLoS Genetics : a peer-reviewed, open-access journal}, volume = {9}, journal = {PLoS Genetics : a peer-reviewed, open-access journal}, number = {8}, publisher = {PLoS}, address = {San Fransisco}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1003767}, pages = {9}, year = {2013}, abstract = {Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacylends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3'-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP): tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.}, language = {en} } @article{VarshneyKumarIgnatovaetal.2012, author = {Varshney, Nishant Kumar and Kumar, R. Suresh and Ignatova, Zoya and Prabhune, Asmita and Pundle, Archana and Dodson, Eleanor and Suresh, C. G.}, title = {Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis}, series = {Acta crystallographica : Section F, Structural biology communications}, volume = {68}, journal = {Acta crystallographica : Section F, Structural biology communications}, number = {3}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {1744-3091}, doi = {10.1107/S1744309111053930}, pages = {273 -- 277}, year = {2012}, abstract = {The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C2221, with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 angstrom, and P41212, with unit-cell parameters a = b = 85.6, c = 298.8 angstrom. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the beta-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme.}, language = {en} } @article{RoethleinMiettinenBorwankaretal.2014, author = {Roethlein, Christoph and Miettinen, Markus S. and Borwankar, Tejas and Buerger, Joerg and Mielke, Thorsten and Kumke, Michael Uwe and Ignatova, Zoya}, title = {Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay}, series = {The journal of biological chemistry}, volume = {289}, journal = {The journal of biological chemistry}, number = {39}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M114.581991}, pages = {26817 -- 26828}, year = {2014}, abstract = {The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered beta-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure.}, language = {en} } @article{RoethleinMiettinenIgnatova2015, author = {R{\"o}thlein, Christoph and Miettinen, Markus S. and Ignatova, Zoya}, title = {A flexible approach to assess fluorescence decay functions in complex energy transfer systems}, series = {BMC biophysics}, volume = {8}, journal = {BMC biophysics}, publisher = {BioMed Central}, address = {London}, issn = {2046-1682}, doi = {10.1186/s13628-015-0020-z}, pages = {10}, year = {2015}, abstract = {Background: Time-correlated Forster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions. Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems. Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems.}, language = {en} } @article{AdamlaIgnatova2015, author = {Adamla, Frauke and Ignatova, Zoya}, title = {Somatic expression of unc-54 and vha-6 mRNAs declines but not pan-neuronal rgef-1 and unc-119 expression in aging Caenorhabditis elegans}, series = {Scientific reports}, volume = {5}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/srep10692}, pages = {10}, year = {2015}, abstract = {Aging is a highly controlled biological process characterized by a progressive deterioration of various cellular activities. One of several hallmarks of aging describes a link to transcriptional alteration, suggesting that it may impact the steady-state mRNA levels. We analyzed the mRNA steady-state levels of polyCAG-encoding transgenes and endogenous genes under the control of well-characterized promoters for intestinal (vha-6), muscular (unc-54, unc-15) and pan-neuronal (rgef-1, unc-119) expression in the nematode Caenorhabditis elegans. We find that there is not a uniform change in transcriptional profile in aging, but rather a tissue-specific difference in the mRNA levels of these genes. While levels of mRNA in the intestine (vha-6) and muscular (unc-54, unc-15) cells decline with age, pan-neuronal tissue shows more stable mRNA expression (rgef-1, unc-119) which even slightly increases with the age of the animals. Our data on the variations in the mRNA abundance from exemplary cases of endogenous and transgenic gene expression contribute to the emerging evidence for tissue-specific variations in the aging process.}, language = {en} } @article{HessSaffertLiebetonetal.2015, author = {Hess, Anne-Katrin and Saffert, Paul and Liebeton, Klaus and Ignatova, Zoya}, title = {Optimization of Translation Profiles Enhances Protein Expression and Solubility}, series = {PLoS one}, volume = {10}, journal = {PLoS one}, number = {5}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0127039}, pages = {14}, year = {2015}, abstract = {mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.}, language = {en} } @inproceedings{FerrolinoZhuravlevaIgnatovaetal.2012, author = {Ferrolino, Mylene and Zhuravleva, Anastasia and Ignatova, Zoya and Gierasch, Lila}, title = {Exploring In vitro and in vivo aggregation of a beta-Clam protein}, series = {Protein science : a publication of the Protein Society}, volume = {21}, booktitle = {Protein science : a publication of the Protein Society}, number = {2}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0961-8368}, pages = {89 -- 89}, year = {2012}, language = {en} } @article{VallerianiZhangNagaretal.2011, author = {Valleriani, Angelo and Zhang, Gong and Nagar, Apoorva and Ignatova, Zoya and Lipowsky, Reinhard}, title = {Length-dependent translation of messenger RNA by ribosomes}, series = {Physical review : E, Statistical, nonlinear and soft matter physics}, volume = {83}, journal = {Physical review : E, Statistical, nonlinear and soft matter physics}, number = {4}, publisher = {American Physical Society}, address = {College Park}, issn = {1539-3755}, doi = {10.1103/PhysRevE.83.042903}, pages = {4}, year = {2011}, abstract = {A simple measure for the efficiency of protein synthesis by ribosomes is provided by the steady state amount of protein per messenger RNA (mRNA), the so-called translational ratio, which is proportional to the translation rate. Taking the degradation of mRNA into account, we show theoretically that both the translation rate and the translational ratio decrease with increasing mRNA length, in agreement with available experimental data for the prokaryote Escherichia coli. We also show that, compared to prokaryotes, mRNA degradation in eukaryotes leads to a less rapid decrease of the translational ratio. This finding is consistent with the fact that, compared to prokaryotes, eukaryotes tend to have longer proteins.}, language = {en} } @article{BorwankarRoethleinZhangetal.2011, author = {Borwankar, Tejas and Roethlein, Christoph and Zhang, Gong and Techen, Anne and Dosche, Carsten and Ignatova, Zoya}, title = {Natural osmolytes remodel the aggregation pathway of mutant huntingtin exon 1}, series = {Biochemistry}, volume = {50}, journal = {Biochemistry}, number = {12}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/bi1018368}, pages = {2048 -- 2060}, year = {2011}, abstract = {In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types to regulate the intracellular solvent quality and to counteract the deleterious effect on the stability and function of cellular proteins. Given the evidence that destabilization of the native state of a protein either by mutation or by environmental changes triggers the aggregation in the neurodegenerative pathologies, the modulation of the intracellular solute composition with osmolytes is an attractive strategy to stabilize an aggregating protein. Here we report the effect of three natural osmolytes on the in vivo and in vitro aggregation landscape of huntingtin exon 1 implicated in the Huntington's disease. Trimethylamine N-oxide (TMAO) and proline redirect amyloid fibrillogenesis of the pathological huntingtin exon 1 to nonamyloidogenic amorphous assemblies via two dissimilar molecular mechanisms. TMAO causes a rapid formation of bulky amorphous aggregates with minimally exposed surface area, whereas proline solubilizes the monomer and suppresses the accumulation of early transient aggregates. Conversely, glycine betaine enhances fibrillization in a fashion reminiscent of the genesis of functional amyloids. Strikingly, none of the natural osmolytes can completely abrogate the aggregate formation; however, they redirect the amyloidogenesis into alternative, nontoxic aggregate species. Our study reveals new insights into the complex interactions of osmoprotectants with polyQaggregates.}, language = {en} }