Small-Molecule Inhibitors of Hif-2a Translation Link, Article Critique Example

Small-Molecule Inhibitors of HIF-2a Translation Link Its 50UTR Iron-Responsive Element to Oxygen Sensing

A brief summary of the background

Researchers in this study carried this study with the prior knowledge that hypoxia affects individuals by altering the rate and nature of protein translation. With the unclear mechanisms in how the body can be able to escape such occurrences, Zimmer et al screened and identified minute molecules, which functioned to limit the existence and translation of HIF-2a. With the subsequent effect on IRP1 and IRE, they held to the fact that the levels of HIF-2a were significantly decreased and suppressed by hypoxia. Eventually, the researchers had to carry out an analysis seeking to describe how translation mechanism of HIF-2a enhanced messaged retention in hypoxic conditions by inhibiting IRP1. In their study, the researchers also sought to provide the relevant tools that were necessary for executing this study successfully.

It is observed that the body tries to conserve energy during hypoxic conditions by altering the translation of proteins. In such a case, the binding of DNA to the target site are altered through dimerization. As a result, the researchers had to develop a method of screening cells possessing HIF inhibitors and those with multiple cancer cell lines. During the study, they realized that HIF-2a with UTR were enough compounds that could elicit the sensitivity of various compounds. They then carried out some deletion analysis of 5UTR and realized that the limited amount of IRE was required to confer inhibition. With subsequent EMSAs, it was found out that some elements promoted the binding of IRP1 to HIF-2a.

It is evident that there are numerous health hazard associated with hypoxia especially on the tissues of the victims. Through the study, there was a need to determine the biological background of it effects on proteins and other facts that were not known. The unclear occurrences about hypoxia appear to have been motivational tool behind this research. With the prior knowledge that they had in this field, it was possible to carry out a focused research, which could demystify some aspects of hypoxia that were not known especially on the effects of protein translation and iron-binding elements. It was also possible to know some of the adaptive aspects that enabled victims survived even in the peak of hypoxia attacks.

A discussion of the results and experiments

From the results that they obtained, HighThorough-put screen for small HIF-2a inhibitors were used, and it was necessary to authenticate HRE with luciferase constructs. When they were mixed with cells lacking VHL, it was possible to screen the extracts from five possible unique libraries including Chembridge and CEREP. From the screens, the researchers identified eight unique or functional compounds that they believed could have the effect of limiting the existence and action of luciferase. Conversely, the same compounds had no observable effect on transfected luciferase reporters. In the dose response curves, it was crucial to treat cells with D and M for two days whilst bringing any action of luciferase to normal. As indicated by the curve, it is evident that it is possible to analyze the actions of the compound used comparatively.

The other relevant experiment was the profiling of genes that had a significant effect on HIF expression. In this case, it was essential to extract RNA from cells with reconstituted VHL, HIF-2a and pTR. From here, it was possible to identify the target genes of the HIF-2a with the help of GSEA algorithm. Through the same mechanism, it was possible to identify cells were affected by compounds like VHL. Eventually, compounds responsible for upregulation and downregulation were identified. Consequently, hierarchal clustering identified possible gene expression that could be reproduced from the isolated profiles.

The other experiment was the in vitro treatment of compound treated cells from HUVEC growth. In this case, the researchers sought to determine the effect of such cells on the proliferation of human umbilical cord. Evidently, the application of the mentioned compound limited the ability of the culture to induce the HUVEC. However, the compounds should no effect at all. In another experiment, it was illustrated that most compounds had some serious effects on HIF-2a in hypoxic conditions. However, when cells were treated with VHL induction of hypoxic conditions was enhanced. The expression of HIF-2a under Normoxic conditions had similar effects.

The researchers saw the necessity of carrying out some quantitative PCR on the RNAs harvested from cells treated with DMSO. It was discovered that there was no expression of HIF-2a while mRNAs fell in numbers significantly. While determining the nascent effect of the compounds on HIF-2a stability, it was essential to assess the half-life of the protein in the presence of cycloheximine. Studies revealed that there were no effects of HIF-2a on protein stability. Consequently, an experiment to determine the effect of mRNA translation affected by various compounds was carried out. The researchers discovered that all the compounds used played a critical role in limiting the amount of HIF-2a protein synthesized. This obviously shows that there was the effect on the occurrence of hypoxia because of the alteration of translation mechanisms.

The other noteworthy experiment was the generation of stable clones that expressed luciferase reporters. It was essential to study the transferin receptor in order to establish the nature of compounds that affected the performance f IRP. In this case, it was established that IRP1 and IRP2 bonded to TfR1 thus altering the stability of mRNA. Further establishments show that mRNA translation was affected appreciably. This is because of the existence of a certain target gene whose messaging limit the expression of cells with VHL. Additionally, inhibitors that bind to HIF also contribute to the instability of mRNA and subsequent translation. Another experiment sought to show compounds that acted courtesy of iron chelation. With the help of EDHB, the absorbance of an ester often used in chelating showed no evidence of effect on mRNA translation. Presence of such compounds were seen to be distinguished stabilizers and activators of HIF.

In the determination of the effect of IRP1 on hypoxic depression, wild types of HIF-2a were generated on luciferase cell lines. When IRP was depleted, it was observed that IRP2 were significantly enhanced in conditions of depression. In the existence of hypoxic and normoxic conditions, it was evident that the translation of HIF-2a were negatively affected.

It is evident from the experiments that compounds which increased hypoxic conditions function well by limiting the ability of IRP1 to bind to HIF-2a. This occurs essentially because the mRNA binding is altered resulting in the depletion of IRP1 and IRP2. This plays role in altering the occurrence of hypoxic conditions in an individual.

A summary of the main conclusions drawn by the authors

Various conclusions can be drawn from the research carried out by the researchers. The research has clearly shown that relying on HIF-2a analysis is fundamental in determining the occurrence of hypoxia among vulnerable individuals. By altering the stability of HIF, it is observed that hypoxia has the tendency of inhibiting the expression and translation proteins, which are crucial in tackling, is occurrence. The researchers have also pointed out the importance of environmental factors in tackling cellular hypoxia. In this case, cells translate efficiently when responsible genes are made enabling the cells to respond as expected. Other conclusions drawn by the researchers is the identification of HIF-2a as the crucial factor in correcting any errors generated by hypoxia. This is supported by the data collected from their study especially on the binding of IRP to HIF-2a. It is evident that hypoxia affects the production of HIF-2a and the subsequent ability to of its binding to mRNA. However, this goes against the existing data and justified their need to use biochemical assays to support the study.

From the study, it is reported that hypoxia has the effect of causing permanence of IRP2 and subsequent fastening to HIF-2a. Additionally, the researchers showed the effect of IRP as the solid regulator to IRE in renal cells. The compounds used are seen to have effect on the ability of HIF-1a to express itself on cells. However, there is varied sensitivity of the compounds have diverse effects on both IRE and HIF-2a. This led them to make the conclusion that the compounds used had a direct correlation in affecting HIF-2a independently. Essentially, the mechanisms involved in the instruction of HIF in hypoxic conditions are rather mild, and no succinct conclusions can be drawn. The role of gene in hypoxic conditions cannot be underestimated especially in the alteration of transcriptional factors like mRNAs.

It is conclusive to draw the following facts from the following study on hypoxia and related compounds. First, HIF transformation are inhibited in instances where IRE exists. The study also identified significant agents, which are useful in tackling the existence of hypoxia in a given population. The approach used in the analysis of genes provides succinct understanding on the way transcription of HIF are mediated and the subsequent retort. It is essential to that into consideration the aspect of mutation through the reorganization of chromatin and the processes that follows mRNA transcription. In the course of transcription, HIF activity is modulated courtesy of the transcriptional factors that also play a critical role in hypoxic conditions. Of considerable interest is the psychology of the victim suffering from hypoxia. Further studies that have to be carried out should focus much in elucidating the role of iron in tackling hypoxia with the help of IRP1. It is obvious that studies relating to human health are infinite, and studies should be continuous. This is the only way that will help other scholars and researchers to determine accurate methods eventually of dealing with hypoxia. This calls for participation of all stakeholders in the medicine field. Evidently, it is necessary to point out the cost of most medical experiments carried out. This requires the government and donors to provide enough funding for the realization of such dreams. This will be helpful to the entire society especially the individuals affected with this menace.