What is protein nitration?

Proteins catalyze a wide range of reactions in the cell and play important structural roles. Post-translational modification of proteins, which includes the alteration of certain amino acid side chains by the introduction of different chemical groups, takes place in eukaryotic and prokaryotic cells. Protein nitration is one example that takes place on tyrosine residues which can have important consequences for the regulation of biological processes. For example, it may result in changes to cell metabolism or lead to alterations in crucial signaling events. The introduction of a nitro- group into a protein molecule is also implicated in different diseases including cardiovascular disease, lung disease, diabetes, cancer and some neurodegenerative conditions.

In this blog, we look at some of the ways a microplate reader can be used to advance protein nitration and related research in the laboratory.

Protein tyrosine nitration and disease

Protein nitration is specifically the introduction of a covalently bound nitro group (NO₂) into a protein. The nitration of proteins can significantly modify both the structural and catalytic roles of these molecules. ^1,2 Protein nitration is a post-translational modification that occurs in both prokaryotic and eukaryotic cells primarily on tyrosine residues.^3,4

Oxidative modifications, including nitration, impact the function of proteins, emphasizing the importance of identifying these modifications for understanding their potential biological implications. 

For example, manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, can undergo nitration of its tyrosine residues, leading to enzyme inactivation and contributing to oxidative stress and related diseases.

While protein nitration is linked to disease, in many cases its precise physiological function is often unknown. Researchers are therefore interested in finding out the implications of these types of chemical alterations for different biological processes to promote the discovery and development of clinical and other applications.

Protein nitration assays on microplate readers 

There are several ways researchers can use microplate readers to study nitrated proteins both direct and indirect. In addition, there are many related assays which help to build context around how protein nitration and processes like nitrosative stress contribute to different biological mechanisms, including signaling events and other processes in healthy and diseased cells.

Enzyme-linked immunosorbent assays (ELISAs) for 3-nitrotyrosine, direct absorbance measurements, fluorescence-based assays and in some cases high throughput screening for inhibitors of protein nitration are all possible and benefit from use of a microplate reader. Detecting nitrated tyrosine residues in these assays presents challenges due to the complexities in accurately quantifying and identifying these specific modifications in proteins. Protein nitration assays are widely used in many research areas including but not limited to biochemistry, biotechnology, cell biology, microbiology, and molecular biology.

Complementary protein nitration assays

Measurements for inducers of protein nitration in cells, such as peroxynitrite, are often encountered in cancer as well as in food research. Peroxynitrite arises from oxidation of nitric oxide in the cell by reactive oxygen species like superoxide. It has been linked to the onset of several diseases and some examples are provided in the next section. In this context, microplate readers also provide an important resource to perform complementary biological assays that build a wider picture of the levels of different biological molecules that are involved in the highly interconnected processes that lead to specific protein nitration. 

Some examples of applications related to protein nitration assays 

Peroxynitrite and protein nitration

In the application note Production of peroxynitrite by cancer-associated immune cells can be detected with a fluorescence-based assay researchers were able to detect peroxynitrite during phagocytosis using a small molecule sensor. As mentioned earlier, peroxynitrite is a reactive oxidant that can act as a nitrating metabolite. It is formed from the reaction of a superoxide radical with nitric oxide, leading to the nitration of tyrosine residues. 

Increased peroxynitrite production by myeloid derived suppressor cells has been observed in the tumor microenvironment of many cancer patients. This complex network of molecules, cells, and structures around a tumor, is of interest to researchers looking for new therapeutic approaches to cancer since it significantly impacts disease progress, how cancer spreads, and the efficacy of treatments. While the effects of peroxynitrite within the tumor microenvironment are not yet fully understood, it seems to contribute to immunosuppression and the protection of cancer from the immune system. 

In the highlighted study, the CLARIOstar^® Plus was used to detect peroxynitrite during phagocytosis with a small molecule sensor. The assay (Fig. 1) made use of a non-fluorescent probe that becomes fluorescent when it reacts with peroxynitrite.

The RAW264.7 macrophage cell line used in the study showed fluorescence only when conditions for antibody-dependent cellular phagocytosis were met. Similarly, the myeloid-derived suppressor cell (MDSC)-like cell line MSC2 exhibited increased fluorescence under conditions where phagocytosis was stimulated (Fig. 2). The robust fluorescent signal detected in MSC2 cells encouraged evaluation of this assay for the detection of peroxynitrite in mouse models of cancer as well as human cancer patients where MDSC expression was expanded.

Nitrosative stress and protein nitration

Nitrosative stress and protein nitration are thought to be linked to disease as part of a complex interplay between several molecular processes. The physicochemical and biological consequences of protein tyrosine nitration can significantly affect protein function and cellular processes, leading to alterations in biochemical reactions and biological pathways. The study Nitration of Hsp90 induces cell death for example describes how protein nitration as a result of oxidative stress can turn a normally helpful protein into a toxic protein.⁵ This 90-kDa heat-shock protein is a part of a highly conserved family of proteins that are well characterized for their function as chaperones. At first, it may seem surprising for Hsp 90 to have the ability to convert into a protein that induces cell death. However, Hsp 90 plays a role in up to 200 cell functions. If it is shut down by tyrosine protein nitration, all of those cell functions are adversely affected.

Some studies have identified a link between mitochondrial stress, the levels of protein nitration, and disease pathology in neurological disease. In these cases, it is useful not only to measure different small molecules (e.g. reactive oxygen or nitrogen species) but also the specific enzymes or mutated enzymes that might be the target of protein nitration through nitrosative-related or oxygen-related stress. Significantly, increased production of reactive-oxygen species often takes place with the parallel accumulation of reactive nitrogen species.

In the paper Mitochondrial oxidant stress promotes α-synuclein aggregation and spreading in mice with mutated glucocerebrosidase researchers used fluorescence assays on a BMG LABTECH Omega series microplate reader to examine the impact of these different stress factors on the activity of the lysosomal enzyme glucocerebrosidase, mutations of which have been linked to certain types of Parkinson’s disease.⁶ 4-Methylumbelliferone, the product of enzymatic glucocerebrosidase activity, was used as readout for this assay. Samples were assayed in duplicates and quantified using fluorescence detection (365 nm excitation and 450 nm emission). (Fig. 3). The study provided evidence of a mechanistic link between expression of mutated enzyme, mitochondrial reactive oxygen species burden, nitrosative stress, and exacerbation of aggregation and interneuronal brain transfer of alpha-synuclein.

In the paper Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer researchers needed to measure a wide range of parameters related to nitrosative stress and protein nitration.⁷ This included nitrite and nitrate measurements (Fig. 4), nitrosothiol levels, changes in cGMP (Fig. 4), alterations in glutathione, as well as ELISA determinations of the levels of green fluorescent protein as a reporter of nitrosative stress. All these assays were performed using either absorbance-based or fluorescence-based assays on an Omega series microplate reader.

In this way, the researchers demonstrated that cerebral disease in argininosuccinic aciduria involves neuronal oxidative and nitrosative stress independent of hyperammonaemia (a disease that gives rise to elevated ammonia levels in the blood). They also showed that neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Going one step further, they demonstrated that disease amelioration by simultaneous brain and liver gene transfer with one vector was possible in mice to treat both metabolic pathways involved [hepatic urea cycle detoxifying ammonia and the citrulline-nitric oxide cycle producing NO], work which provides new hope for patients with hepatocerebral metabolic diseases.

In the paper S100B Protein but Not 3-Nitrotyrosine Positively Correlates with Plasma Ammonia in Patients with Inherited Hyperammonemias: A New Promising Diagnostic Tool? a group of researchers used ELISA assays performed on a SPECTROstar^® Nano to determine plasma levels of 3-nitrotyrosine.8 A second sandwich ELISA with absorbance detection allowed measurement of the concentrations of the S100 calcium-binding protein (S100B). Advanced oxidation protein products were also determined (Fig. 5).

The study documented two key findings: a positive correlation between ammonia and S100B levels; and distinctive levels of 3-nitrotyrosine that did not correlate with ammonia in the plasma of studied patients. The work showed that S100B and 3-nitrotyrosine may have diagnostic utility in patients with congenital hyperammonemias. These molecules may serve as circulating indicators of neurological decline and systemic oxidative–nitrosative stress, respectively, in the blood of patients.

Assays for reactive nitrogen species other than those involved in protein nitration 

Post-translational modifications can lead to significant changes in protein structure, affecting both the physical arrangement of amino acids and the biochemical activities within biological systems. Protein nitration is not the only type of post-translational modification of proteins. The presence of sulfhydryl groups is another example. In the application note Comparison of thioredoxin activity in cortical neuron and glial cells using a BMG LABTECH microplate reader a thioredoxin system that contains sulfhydryl groups protected cells against H₂O₂-induced cell death and its inhibition promoted oxidative stress, while thioredoxin-overexpressing mice displayed less oxidative brain damage following ischemia and lived longer.

Conclusion

Much remains to be discovered about how protein nitration contributes to healthy cells and its link to disease. Protein nitration can significantly impact protein function by altering the 3D structure, binding capabilities, stability, and localization of proteins, which may influence biological functions and signaling pathways in many ways, particularly under stress conditions. The diversity of assays that can be performed on microplates and the efficiencies microplate readers bring to the measurements needed for these assays should advance discoveries and in the longer term should help provide clinical benefits for unmet needs in several disease areas.

BMG LABTECH solutions

What is the preferred BMG LABTECH microplate reader for specific needs and applications for protein nitration?

As highlighted above, assays revolving around protein nitration are mainly focused on absorbance and fluorescence readouts. Accordingly, all BMG LABTECH multi-mode microplate readers are suited for performing these applications, while the SPECTROstar Nano is a viable option for exclusively performing absorbance-based protein nitration assays. 

All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series and PHERAstar^® FSX microplate readers come complete with on-board injectors that can offer the very best options for detection at the time of injection.

Both the VANTAstar^® and CLARIOstar^® Plus allow for fluorescence emission scanning and include Enhanced Dynamic Range technology for superior performance in a single run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators and different filter options offering maximal flexibility in the setup of everyday applications as well as specific protein nitration assays. 

The PHERAstar FSX is the option of choice if researchers need the highest possible sensitivity and the fastest speed at scale for their protein folding or conformational state measurements. It was specifically conceived for screening campaigns and offers the level of performance suitable for high-throughput investigations.

Collectively, these multi-mode readers combine high-quality measurements with miniaturized assays, short measurement times, and offer considerable savings on materials and other resources.

Configure your microplate reader and get an initial recommendation!

Configure

References

1. Bottari SP. Protein tyrosine nitration: a signaling mechanism conserved from yeast to man. Proteomics. 2015 Jan;15(2-3):185-7. doi: 10.1002/pmic.201400592.
2. Radi R. Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4003-8. doi: 10.1073/pnas.0307446101. Epub 2004 Mar 12.
3. Lee JR, Kim JK, Lee SJ, Kim KP. Role of protein tyrosine nitration in neurodegenerative diseases and atherosclerosis. Arch Pharm Res. 2009 Aug;32(8):1109-18. doi: 10.1007/s12272-009-1802-0.
4. Peluffo G, Radi R. Biochemistry of protein tyrosine nitration in cardiovascular pathology. Cardiovasc Res. 2007 Jul 15;75(2):291-302. doi: 10.1016/j.cardiores.2007.04.024.
5. Franco MC, Ye Y, Refakis CA, Feldman JL, Stokes AL, Basso M, Melero Fernández de Mera RM, Sparrow NA, Calingasan NY, Kiaei M, Rhoads TW, Ma TC, Grumet M, Barnes S, Beal MF, Beckman JS, Mehl R, Estévez AG. Nitration of Hsp90 induces cell death. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):E1102-11. doi: 10.1073/pnas.1215177110. Epub 2013 Mar 4.
6. La Vitola P, Szegö EM, Pinto-Costa R, Rollar A, Harbachova E, Schapira AH, Ulusoy A, Di Monte DA. Mitochondrial oxidant stress promotes α-synuclein aggregation and spreading in mice with mutated glucocerebrosidase. NPJ Parkinsons Dis. 2024 Dec 11;10(1):233. doi: 10.1038/s41531-024-00842-8. Fig.3 in this blog adapted from this reference under license CC-BY 4.0.
7. Baruteau J, Perocheau DP, Hanley J, Lorvellec M, Rocha-Ferreira E, Karda R, Ng J, Suff N, Diaz JA, Rahim AA, Hughes MP, Banushi B, Prunty H, Hristova M, Ridout DA, Virasami A, Heales S, Howe SJ, Buckley SMK, Mills PB, Gissen P, Waddington SN. Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer. Nat Commun. 2018 Aug 29;9(1):3505. doi: 10.1038/s41467-018-05972-1. Fig.4 in this blog adapted from this reference under license CC-BY 4.0.
8. Czarnecka AM, Obara-Michlewska M, Wesół-Kucharska D, Greczan M, Kaczor M, Książyk J, Rokicki D, Zielińska M. S100B Protein but Not 3-Nitrotyrosine Positively Correlates with Plasma Ammonia in Patients with Inherited Hyperammonemias: A New Promising Diagnostic Tool? J Clin Med. 2023 Mar 21;12(6):2411. doi: 10.3390/jcm12062411. jFig.5 in this blog adapted from this reference under license CC-BY 4.0.