104 research outputs found
Folding Circular Permutants of IL-1β: Route Selection Driven by Functional Frustration
Interleukin-1β (IL-1β) is the cytokine crucial to inflammatory and immune response. Two dominant routes are populated in the folding to native structure. These distinct routes are a result of the competition between early packing of the functional loops versus closure of the β-barrel to achieve efficient folding and have been observed both experimentally and computationally. Kinetic experiments on the WT protein established that the dominant route is characterized by early packing of geometrically frustrated functional loops. However, deletion of one of the functional loops, the β-bulge, switches the dominant route to an alternative, yet, as accessible, route, where the termini necessary for barrel closure form first. Here, we explore the effect of circular permutation of the WT sequence on the observed folding landscape with a combination of kinetic and thermodynamic experiments. Our experiments show that while the rate of formation of permutant protein is always slower than that observed for the WT sequence, the region of initial nucleation for all permutants is similar to that observed for the WT protein and occurs within a similar timescale. That is, even permutants with significant sequence rearrangement in which the functional-nucleus is placed at opposing ends of the polypeptide chain, fold by the dominant WT “functional loop-packing route”, despite the entropic cost of having to fold the N- and C- termini early. Taken together, our results indicate that the early packing of the functional loops dominates the folding landscape in active proteins, and, despite the entropic penalty of coalescing the termini early, these proteins will populate an entropically unfavorable route in order to conserve function. More generally, circular permutation can elucidate the influence of local energetic stabilization of functional regions within a protein, where topological complexity creates a mismatch between energetics and topology in active proteins
Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection
cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu).Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work.Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.Peer Reviewe
Forty years on: clathrin-coated pits continue to fascinate
Clathrin mediated endocytosis (CME) is a fundamental process in cell biology and has been extensively investigated throughout the last several decades. Every cell biologist learns about it at some point during their education and the beauty of this process has led many of us to go deeper and make it the topic of our own research. Great progress has been made towards elucidating the mechanisms of CME and the field is becoming increasingly complex with several hundred new publications every year. This makes it easy to get lost in the vast amount of literature and to forget about the fundamentals of the field, based on the careful interpretation of simple observations made over 40 years ago. A study performed by Anderson, Brown and Goldstein in 1977 (Anderson et al., 1977) is a prime example of this. We therefore want to take a step back and examine how this seminal study was pivotal to our understanding of CME and its progression into ever increasing complexity over the last four decades
Exploring Off-Targets and Off-Systems for Adverse Drug Reactions via Chemical-Protein Interactome — Clozapine-Induced Agranulocytosis as a Case Study
In the era of personalized medical practice, understanding the genetic basis of patient-specific adverse drug reaction (ADR) is a major challenge. Clozapine provides effective treatments for schizophrenia but its usage is limited because of life-threatening agranulocytosis. A recent high impact study showed the necessity of moving clozapine to a first line drug, thus identifying the biomarkers for drug-induced agranulocytosis has become important. Here we report a methodology termed as antithesis chemical-protein interactome (CPI), which utilizes the docking method to mimic the differences in the drug-protein interactions across a panel of human proteins. Using this method, we identified HSPA1A, a known susceptibility gene for CIA, to be the off-target of clozapine. Furthermore, the mRNA expression of HSPA1A-related genes (off-target associated systems) was also found to be differentially expressed in clozapine treated leukemia cell line. Apart from identifying the CIA causal genes we identified several novel candidate genes which could be responsible for agranulocytosis. Proteins related to reactive oxygen clearance system, such as oxidoreductases and glutathione metabolite enzymes, were significantly enriched in the antithesis CPI. This methodology conducted a multi-dimensional analysis of drugs' perturbation to the biological system, investigating both the off-targets and the associated off-systems to explore the molecular basis of an adverse event or the new uses for old drugs
IL-1 Receptor Antagonist Blocks the Lipopolysaccharide-Induced Inhibition of Gastric Motility in Freely Moving Conscious Rats
Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation
- …