Molecular forms of IGF binding protein 2 and their presence in various pathophysiological states

Везујући протеин 2 за факторе раста сличне инсулину (IGFBP-2) у циркулацији се може јавити у три облика: као комплекс, мономер и фрагмент различитих молекулских маса. У оквиру ове докторске дисертације је утврђено да IGFBP-2 гради комплексе са α-2-макроглобулином (α2М).Релативни удео комплекса IGFBP-2/α2М у укупном IGFBP-2 не зависи директно од концентрације IGFBP-2 и α2М, већ зависи од различитих (пато)физиолошкихуслова у којима се организам налази. У овом раду су испитане различите методе за изоловање, мерење и карактеризацију комплекса IGFBP-2/α2М. Такође је испитана промена и могућа улога комплекса код пацијената са тумором.Познато је да мономер IGFBP-2 везује IGF лиганде са великим афинитетом и транспортује их до ткива где се, након протеолизе IGFBP-2, отпуштају и везују за специфичне ћелијске рецепторе. Осим као носач IGF пептида, IGFBP-2 испољава и независна метаболичка и митогена дејства. Везујући се за интегринске рецепторе (првенствено за α5β1), IGFBP-2 стимулише покретљивост ћелије и њено одвајање од околине, доприносећи метастатском потенцијалу. Неки фрагменти могу слабо везати IGF лиганде и интераговати са ћелијама. О комплексима IGFBP-2 у циркулацији до сада није било података у литератури.У раду је показано да се врста молекулских облика IGFBP-2 не мења под утицајем различитих (пато)физиолошких фактора, као што су старење, активно бављење спортом, оксидативни стрес, повећанa концентрацијa липида или глукозе, измењена протеолитичка активност, али се мења њихова количина и међусобни однос. Старењем се повећава концентрација мономера и фрагмената IGFBP-2 у циркулацији, као и α2М, а смањује се концентрација комплекса IGFBP-2/α2М. Јони цинка (II) подстичу олигомеризацију α2M, али не утичу на стварање комплекса. Пептидна секвенца RGD, која је важна за интеракцију IGFBP-2 са интегрином, није контактна секвенца за интеракцију IGFBP-2 са α2М.Код пацијената са тумором дебелог црева је измерена повећана концентрација укупног IGFBP-2 у циркулацији у односу на здраве људе, као и измењен међусобни однос молекулских форми. Повећана је концентрација мономера и фрагмената, а смањена комплекса. У ткиву дебелог црева су нађени само мономер и фрагменти...In circulation, insulin-like growth factor binding protein 2 (IGFBP-2), can be found in three main forms: as a complex, monomer and assembley of fragments of differents molecular masses. In making this dissertation it was found that IGFBP-2 forms complexes with α-2-macroglobulin (α2M).Relative amount of IGFBP-2/α2M complex in total IGFBP-2 concentration does not depend on concentrations of IGFBP-2 and α2M, but from various (patho)physiological conditions in organism. In this work, different methods for isolation, measurement and characterisation of IGFBP-2/α2M complex were examined. An investigation on potential role of these complexes in patients with tumor was also conducted.It is known that IGFBP-2 monomer binds IGF ligands with high affinity and transports them to tissues where, after proteolysis, they are released, and bound to specific receptors. Except being the IGF carrier, IGFBP-2 exerts IGF-independent metabolic and mitogenic actions. It can bind to integrin receptors (primarily to α5β1) and stimulate cell motility and detachement from their surroundings, contributing to metastatic potential. Some fragments can loosely bind IGF ligands and interact with cells. Until know, there was no literature data about IGFBP-2 complexes in circulation.In this work, it was shown that the distribution of molecular species of IGFBP-2 does not change under the influence of different (patho)physiological factors, such as aging, intensive physical activity, oxidative stress, increased concentration of lipids and glucose, impaired proteolytic activity, but by the quantity and their mutual ratio change. With aging, the concentration of IGFBP-2 monomers and fragments, and α2M, in circulation increases, while the concentration of IGFBP-2/α2M complex decreases. Zinc ions encourage the α2M oligomerisation, but have no influence on complex formation. RGD peptide sequence, which is important for IGFBP-2 interaction with integrins, is not a contact sequence for interaction between IGFBP-2 and α2M.In serum of patients with colon cancer, increased concentration of IGFBP-2 was detected, as well as different relation of molecular forms. The concentration of monomer and fragments increased, while the concentration of complexes decreased..

Molecular forms of IGF binding protein 2 and their presence in various pathophysiological states

Везујући протеин 2 за факторе раста сличне инсулину (IGFBP-2) у циркулацији се може јавити у три облика: као комплекс, мономер и фрагмент различитих молекулских маса. У оквиру ове докторске дисертације је утврђено да IGFBP-2 гради комплексе са α-2-макроглобулином (α2М).Релативни удео комплекса IGFBP-2/α2М у укупном IGFBP-2 не зависи директно од концентрације IGFBP-2 и α2М, већ зависи од различитих (пато)физиолошкихуслова у којима се организам налази. У овом раду су испитане различите методе за изоловање, мерење и карактеризацију комплекса IGFBP-2/α2М. Такође је испитана промена и могућа улога комплекса код пацијената са тумором.Познато је да мономер IGFBP-2 везује IGF лиганде са великим афинитетом и транспортује их до ткива где се, након протеолизе IGFBP-2, отпуштају и везују за специфичне ћелијске рецепторе. Осим као носач IGF пептида, IGFBP-2 испољава и независна метаболичка и митогена дејства. Везујући се за интегринске рецепторе (првенствено за α5β1), IGFBP-2 стимулише покретљивост ћелије и њено одвајање од околине, доприносећи метастатском потенцијалу. Неки фрагменти могу слабо везати IGF лиганде и интераговати са ћелијама. О комплексима IGFBP-2 у циркулацији до сада није било података у литератури.У раду је показано да се врста молекулских облика IGFBP-2 не мења под утицајем различитих (пато)физиолошких фактора, као што су старење, активно бављење спортом, оксидативни стрес, повећанa концентрацијa липида или глукозе, измењена протеолитичка активност, али се мења њихова количина и међусобни однос. Старењем се повећава концентрација мономера и фрагмената IGFBP-2 у циркулацији, као и α2М, а смањује се концентрација комплекса IGFBP-2/α2М. Јони цинка (II) подстичу олигомеризацију α2M, али не утичу на стварање комплекса. Пептидна секвенца RGD, која је важна за интеракцију IGFBP-2 са интегрином, није контактна секвенца за интеракцију IGFBP-2 са α2М.Код пацијената са тумором дебелог црева је измерена повећана концентрација укупног IGFBP-2 у циркулацији у односу на здраве људе, као и измењен међусобни однос молекулских форми. Повећана је концентрација мономера и фрагмената, а смањена комплекса. У ткиву дебелог црева су нађени само мономер и фрагменти...In circulation, insulin-like growth factor binding protein 2 (IGFBP-2), can be found in three main forms: as a complex, monomer and assembley of fragments of differents molecular masses. In making this dissertation it was found that IGFBP-2 forms complexes with α-2-macroglobulin (α2M).Relative amount of IGFBP-2/α2M complex in total IGFBP-2 concentration does not depend on concentrations of IGFBP-2 and α2M, but from various (patho)physiological conditions in organism. In this work, different methods for isolation, measurement and characterisation of IGFBP-2/α2M complex were examined. An investigation on potential role of these complexes in patients with tumor was also conducted.It is known that IGFBP-2 monomer binds IGF ligands with high affinity and transports them to tissues where, after proteolysis, they are released, and bound to specific receptors. Except being the IGF carrier, IGFBP-2 exerts IGF-independent metabolic and mitogenic actions. It can bind to integrin receptors (primarily to α5β1) and stimulate cell motility and detachement from their surroundings, contributing to metastatic potential. Some fragments can loosely bind IGF ligands and interact with cells. Until know, there was no literature data about IGFBP-2 complexes in circulation.In this work, it was shown that the distribution of molecular species of IGFBP-2 does not change under the influence of different (patho)physiological factors, such as aging, intensive physical activity, oxidative stress, increased concentration of lipids and glucose, impaired proteolytic activity, but by the quantity and their mutual ratio change. With aging, the concentration of IGFBP-2 monomers and fragments, and α2M, in circulation increases, while the concentration of IGFBP-2/α2M complex decreases. Zinc ions encourage the α2M oligomerisation, but have no influence on complex formation. RGD peptide sequence, which is important for IGFBP-2 interaction with integrins, is not a contact sequence for interaction between IGFBP-2 and α2M.In serum of patients with colon cancer, increased concentration of IGFBP-2 was detected, as well as different relation of molecular forms. The concentration of monomer and fragments increased, while the concentration of complexes decreased..

The importance of estimation of RAD51 genetic polymorphism in order to predict the occurence and prognosis of colorectal cancer in population in Serbia

Gen RAD 51 igra važnu ulogu u razmeni homologih lanaca u reparaciji DNK. Dva najčešća polimorfizma jednog nukleotida kod ovog gena, 135G˃C i 172G˃T, su povezana sa izmenjenom genskom transkripcijom. Dok je 135G˃C već pov ezan sa karcinomima dojke i kolorektuma, 172G˃T je daleko manje ispitivan, iako sporadične studije pokazuju da bi mogao biti prognostički faktor kod nekih malignih lezija. Cilj ove studije je da se istraži RAD51 172G˃T polimorfizam kod populacije u Srbiji, njegova povezanost sa kolorektalnim karcinomom, kao i korelacija karakteristika bolesti i odgovor na neoadjuvantnu hemioradioterapiju. Metode: Polimorfizam 172G˃T je evaluiran PCR-RFLP metodom iz uzoraka krvi 209 pacijenata sa kolorektalnim karcinomom i 43 zdrava ispitanika koji su služili kao kontrolna grupa. Distribucija genotipova je takođe analizirana u odnosu na određene karakterisike tumora u sluajevima u kojim su histopatološki podaci bili dostupni, kao i u odnosu na dvogodišnje preživljavanje i period do progresije bolesti. Rezultati: Pokazala se značajna povezanost RAD51 172G˃T polimorfizma i dezmoplastične reakcije kolorektalnog karcinoma. Alel 172G je značajno učestaliji kod pacijenata sa intenzivnijim dezmoplastičnim odgovorom na tumorsko tkivo. Nije viđena značajna razlika u preživljavanju niti u periodu do progresije bolesti u grupama sa različitim alelima. Zaključak: rezultati ove studije sugerišu da bi 172T alel gena RAD51 može biti povoljan faktor kod pacijenata sa kolorektalnim karcinomomkod populacije u Srbiji, mada su neophodne veće prospektivne studije da bi se ovaj nalaz potvrdio.The RAD51 gene plays an important role in homologous strand exchange in DNA repair. Two common single nucleotide polymorphisms in this gene, 135G˃C and 172G˃T, were associated with altered gene transcription. While 135G˃C was already linked to breast and colorectal cancers in certain populations, 172G˃T is far less investigated, although sporadic studies showed it could be a prognostic factor for some cancerous lesions. The aim of this study was to investigate RAD51 172G˃T polymorphism in Serbian population, its association with colorectal carcinoma, as well as correlation with disease characteristics and response to neoadjuvant chemoradiotherapy therapy. Methods: The 172G˃T polymorphism was evaluated by PCR-RFLP method in blood samples of 209 colorectal cancer subjects and 43 healthy subjects who served as controls. The distribution of genotypes was also analyzed in respect to several tumor characteristics in cases where histopathological data were available, as well as to verall survival and the disease free interval. Results: A significant association between the RAD51 172G˃T polymoprhism and desmoplastic reaction of colorectal cancer was demonstrated. The 172G allele was found to be significantly more frequent in patients with more intensive desmoplastic response of the tumor tissue. No significant difference was found regarding ovarall survival and the disease free interval. Conclusions: The results of our study suggest that the 172T allele of RAD51 may be a favoring prognostic factor in patients with colorectal cancer in Serbian population, although larger prospective studies are required to confirm this finding

Interaction between alpha-2-macroglobulin and phycocyanobilin – structural and physiological implications

The interaction between phycocyanobilin (PCB), a bioactive chromophore of blue-green cyanobacteria Spirulina’s phycobiliproteins, and human alpha-2-macroglobulin (a2M), a universal anti-proteinase, was investigated in this study under simulated physiological conditions using spectroscopic techniques and a2M activity assay. Protein a2M was found to bind PCB with a moderate affinity, as assessed by spectrofluorimetric titration. The binding constant was calculated to be 6.39105 M 1 at 25°C. The binding of PCB to a2M did not cause significant change in the secondary structure of the protein, as determined by circular dichroism. PCB protected a2M from oxidative damage in the presence of AAPH-induced free radical overproduction. PCB binding also effectively preserved a2M anti-proteinase activity. Since a2M is involved in controlling the action of enzymes during the inflammatory process, the protection that PCB expresses could indirectly influence the intensity and direction of the body response to impaired homeostasis, especially under oxidative stress.The Biochemistry Global Summit, 25th IUBMB Congress, 46th FEBS Congress, 15th PABMB Congress, July 9-14, 2022, Lisbon, Portuga

Interaction between alpha-2-macroglobulin and phycocyanobilin – structural and physiological implications

In this study, the interaction between phycocyanobilin (PCB)1, a bioactive chromophore of blue-green algae Spirulina's phycobiliproteins, and alpha-2-macroglobulin (α2M)2, a universal anti-proteinase, was investigated under simulated physiological conditions using spectroscopic techniques and α2M activity assay. Using spectrofluorimetric measurements, we found that α2M binds PCB with a moderate affinity, with a binding constant of 6.3× 105 M−1 at 25°C. The binding of PCB to α2M does not cause any significant change in the secondary structure of the protein (circular dichroism measurements). Besides, PCB protects α2M from structural oxidative alterations under AAPH-induced free radical overproduction. Further, PCB binding effectively preserves α2M anti-proteinase activity. Since α2M is involved in controlling the action of enzymes during the inflammatory process, the protection that PCB expresses could indirectly influence the intensity and direction of body response to impaired homeostasis, especially under oxidative stress

Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography

Alpha-2-macroglobulin (α2M) is a homotetrameric blood glycoprotein having molecular mass of 720 kDa which acts as a general protease inhibitor 1. So far, the methods to estimate the quantity of α2M and its activity were separate procedures. The quantity is usually measured by immunochemical assays and the anti-protease activity of α2M by measuring the activity of trypsin bound to α2M using chromogenic substrate BAPNA 2. A simple and reliable method for determination of the concentration and function of α2M by zymography was developed. This method is based on the covalent binding of α2M and trypsin followed by non-reducing PAGE and zymography with gelatine incorporated in the electrophoretic gel. The results have shown that α2M binds trypsin in a linear, concentration-dependent manner. The sensitivity of the method is 125 nM with an intraassay coefficient of variation 4.2 %. Freezing of α2M induces its partial denaturation, which can be seen as the reduction in the amount of functional molecule and its reactivity with trypsin. The method was further tested using α2M from patients with an end-stage renal disease who are known to be under an increased oxidative stress and inflammation, which are expected to modify the structure of proteins. Using α2M from these patients, lower affinity of α2M towards trypsin was detected when compaired to α2M isolated from healthy persons. The reported zymographic method enables measurement of α2M taking into consideration both its quantity and function, stressing the importance of determination of the amount of physiologically active molecules and not just their total amount present in the sample. Monitoring of the relation quantity/activity becomes very important when the sample originates from an individual exposed to a stress or with a disease accompanied by post-translational modifications of proteins such as diabetes, renal disease or cancer 3. Presented method also enables determination of α2M in the presence of different modifying chemical substances

Examining fatty acid interactions with Arthrospira platensis-derived C-phycocyanin

Cultured meat requires less land and water and is less polluting, but still costly. The critical challenge in cultivated meat science is identifying and developing bovine serum albumin alternatives as the key component in cell media. Phycobiliproteins (PBPs) from micro- and macroalgae are promising candidates for albumin replacement due to their high abundance and well-known excellent antioxidative and metal-binding activities of covalently attached tetrapyrrole chromophores. Considering the importance of fatty acids (FA) binding by albumin for cell cultivation, the additional prerequisites for developing PBPs as albumin replacement components is their validation for the ability to bind FA. This study aims to examine the ability of C-phycocyanin (C-PC), the major PBP of microalgae Arthrospira platensis, to bind seven fatty acids (stearic, palmitic, oleic, elaidic, linoleic, linolenic and docosahexaenoic acid). For this purpose, we employed various optical spectroscopy techniques (fluorescence, CD, and VIS absorption spectroscopy). The protein fluorescence quenching approach demonstrated FA binding affinities ranging from 0.42 to 2.4 x 105 M−1, with the ability of FA to bind at different sites on C-PC. Fatty acid binding induces substantial changes in the VIS absorption spectra of C-PC, indicating the FA are attached in the vicinity of C-PC chromophores. On the other hand, CD spectroscopy did not show significant effects of FA binding on C-PC secondary structure content. Overall, this study revealed C-PC's significant potential in binding FA, the critical prerequisite to replacing albumin for developing animal-free cell media for meat cultivation

Examining fatty acid interactions with Arthrospira platensis-derived C-phycocyanin

Cultured meat requires less land and water and is less polluting, but still costly. The critical challenge in cultivated meat science is identifying and developing bovine serum albumin alternatives as the key component in cell media. Phycobiliproteins (PBPs) from micro- and macroalgae are promising candidates for albumin replacement due to their high abundance and well-known excellent antioxidative and metal-binding activities of covalently attached tetrapyrrole chromophores. Considering the importance of fatty acids (FA) binding by albumin for cell cultivation, the additional prerequisites for developing PBPs as albumin replacement components is their validation for the ability to bind FA. This study aims to examine the ability of C-phycocyanin (C-PC), the major PBP of microalgae Arthrospira platensis, to bind seven fatty acids (stearic, palmitic, oleic, elaidic, linoleic, linolenic and docosahexaenoic acid). For this purpose, we employed various optical spectroscopy techniques (fluorescence, CD, and VIS absorption spectroscopy). The protein fluorescence quenching approach demonstrated FA binding affinities ranging from 0.42 to 2.4 x 105 M−1, with the ability of FA to bind at different sites on C-PC. Fatty acid binding induces substantial changes in the VIS absorption spectra of C-PC, indicating the FA are attached in the vicinity of C-PC chromophores. On the other hand, CD spectroscopy did not show significant effects of FA binding on C-PC secondary structure content. Overall, this study revealed C-PC's significant potential in binding FA, the critical prerequisite to replacing albumin for developing animal-free cell media for meat cultivation

Dietary fatty acids as a new binding partner of C - phycocyanin: a fluorimetric study

C-Phycocyanin (C-PC) is a phycobiliprotein from cyanobacteria, where it harvests light energy that is then transferred to chlorophylls during photosynthesis. It has an intense blue color due to a covalently bonded tetrapyrrole chromophore, and owing to this property is used in the food industry as a good natural alternative for food coloring. In addition to its coloring properties, C-PC has anti-inflammatory, antioxidant, anti-cancer, and immune-enhancing effects that qualify it as a dietary supplement already included in various formulations, mainly Spirulina extract powders. Since it is used as a food colorant and as a dietary supplement, it may interact with food ingredients, affecting its stability, digestibility, or antioxidant properties. Palmitic acid and linoleic acid (which can be metabolized to linolenic acid) are abundant in meat, milk, and edible oils, so that they could interact with C-PC. C-Phycocyanin isolated from the cyanobacterium Arthrospira platensis (Spirulina) was incubated with increasing concentrations of these three fatty acids, and its fluorescence intensity was monitored. Incubation resulted in a fluorescence quenching effect, indicating that binding had occurred. The binding equations indicated that the association constants were of the same order of magnitude and that the number of approximate binding sites was more than one (Ka = 4.64 x 10⁴ M-¹, n = 1.5 for linoleic acid; Ka = 2.88 x 10⁴ M–¹, n = 1.9 for linolenic acid; Ka = 0.44 x 10⁴ M–¹, n = 0.8 for palmitic acid). This moderate interaction between C-PC and fatty acids could influence its behavior as a nutraceutical and food colorant

Supplementary data for the article: Šunderić, M.; Vasović, T.; Milčić, M.; Miljević, Č.; Nedić, O.; Nikolić, M. R.; Gligorijević, N. Antipsychotic Clozapine Binding to Alpha-2-Macroglobulin Protects Interacting Partners against Oxidation and Preserves the Anti-Proteinase Activity of the Protein. International Journal of Biological Macromolecules 2021, 183, 502–512. https://doi.org/10.1016/j.ijbiomac.2021.04.155.

Supplementary material for: [https://doi.org/10.1016/j.ijbiomac.2021.04.155]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4538