Canine osteosarcoma (OSA) is an aggressive bone neoplasia with high metastatic potential. Metastasis is the main cause of death associated with OSA, and there is no current treatment available for metastatic disease. Proteomic analyses, including matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF/TOF MS), are widely used to select molecular targets and identify proteins that may play a key role in primary tumours and at various steps of the metastatic cascade. The main aim of this study was to identify proteins differently expressed in canine OSA cell lines with different malignancy phenotypes (OSCA-8 and OSCA-32) compared to canine osteoblasts (CnOb). The intermediate aim of the study was to compare canine OSA cell migration capacity and assess its correlation with the malignancy phenotypes of each cell line. Using MALDI-TOF/TOF MS analyses, we identified eight proteins that were significantly differentially expressed (p ≤ 0.05) in canine OSA cell lines compared to CnOb: cilia- and flagella-associated protein 298 (CFAP298), general transcription factor II-I (GTF2I), mirror-image polydactyly gene 1 protein (MIPOL1), alpha-2 macroglobulin (A2M), phosphoglycerate mutase 1 (PGAM1), ubiquitin (UB2L6), ectodysplasin-A receptor-associated adapter protein (EDARADD), and leucine-rich-repeat-containing protein 72 (LRRC72). Using the Simple Western technique, we confirmed high A2M expression in CnOb compared to OSCA-8 and OSCA-32 cell lines (with intermediate and low A2M expression, respectively). Then, we confirmed the role of A2M in cancer cell migration by demonstrating significantly inhibited OSA cell migration by treatment with A2M (both at 10 and 30 mM concentrations after 12 and 24 h) in a wound-healing assay. This study may be the first report indicating A2M\'s role in OSA cell metastasis; however, further in vitro and in vivo studies are needed to confirm its possible role as an anti-metastatic agent in this malignancy.

Alpha-2-macroglobulin (α2-MG) is a multifunctional protein involved in neurodegeneration, inflammation and neovascularization, which are key processes in the pathogenesis of age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). AMD and PDR are two of the main causes of vision loss and blindness, are difficult to treat, and are generally diagnosed at the stage of irreversible changes.
OBJECTIVE: This study estimates the activity of α2-MG in the blood serum and tears of patients with AMD and PDR in order to reveal the relation of its levels with the intensity of the pathological process in the retina.
METHODS: The study included 17 patients (34 eyes) with AMD, 15 patients (30 eyes) with PDR, and 15 healthy adults (30 eyes) of the similar age. The activity of α2-MG in serum and tears was measured enzymatically using the specific substrate N-benzoyl-DL-arginine-p-nitroanilide (BAPNA).
RESULTS: The activity of α2-MG in tears of patients with AMD was on the average 3.5 times higher than in healthy controls, and in patients with PDR - 1.5 times higher. Patients with AMD at the submacular fibrosis stage showed decreased α2-MG activity in tears. The activity of α2-MG in serum of patients with AMD and PDR was on the average 25% higher than in healthy persons. No correlation was revealed between serum and tear levels of α2-MG activity.
CONCLUSIONS: This study revealed for the first time that in AMD and PDR the activity of α2-MG in tears is increased, and that in AMD the increase is higher than in PDR. An increase of α2-MG activity in serum confirms the presence of systemic inflammation. Absence of correlation between the serum and tear activity of α2-MG confirms its local origin. The high level of α2-MG activity in tears reflects the presence of an active destructive process in the retina, justifying its further investigation as a predictor of AMD and PDR course, as well as an indicator of therapy effectiveness.
Многофункциональный белок альфа2-макроглобулин (α2-МГ) принимает участие в развитии нейродегенеративных процессов, воспаления и неоваскуляризации, которые являются ключевыми в патогенезе возрастной макулярной дистрофии (ВМД) и пролиферативной диабетической ретинопатии (ПДР). ВМД и ПДР являются одними из основных причин слабовидения и слепоты, трудно поддаются лечению и диагностируются на стадии необратимых изменений.
UNASSIGNED: Определение активности α2-МГ в слезе и крови пациентов с ВМД и ПДР для выявления связи ее уровня с наличием патологического процесса в сетчатке.
UNASSIGNED: В исследование включено 17 пациентов (34 глаза) с ВМД, 15 пациентов (30 глаз) с ПДР и 15 здоровых добровольцев (30 глаз). В слезе и сыворотке крови определяли активность α2-МГ ферментативным методом со специфическим субстратом N-бензоил-DL-аргинин-p-нитроанилидом.
UNASSIGNED: Активность α2-МГ в слезе пациентов с ВМД была повышена в среднем в 3,5 раза, а у пациентов с ПДР — в 1,5 раза. При ВМД на стадии субмакулярного фиброза активность α2-МГ в слезе была снижена. В крови больных с ВМД и ПДР активность α2-МГ оказалась повышена в среднем на 25%. Корреляции между активностью α2-МГ в слезе и крови не выявлено.
UNASSIGNED: Впервые установлено, что при ВМД и ПДР происходит увеличение активности α2-МГ в слезе, причем при ВМД — более значительное, чем при ПДР. Повышение активности α2-МГ в крови свидетельствует о вялотекущем системном воспалении при ВМД и ПДР. Отсутствие корреляции между активностью α2-МГ в слезе и крови подтверждает его местное происхождение. Высокий уровень α2-МГ в слезе отражает наличие дегенеративного процесса в сетчатке, что обосновывает целесообразность дальнейших исследований α2-МГ для прогнозирования течения ВМД и ПДР, а также контроля терапии.

α2-macroglobulin (α2M) is a versatile endopeptidase inhibitor that plays a role in cell growth, inflammation and coagulation. α2M is an inhibitor of key coagulation enzyme thrombin. Hypercoagulability due to an excess of thrombin production can cause thrombotic events. Therefore, we investigated the association of α2M levels and cardiovascular events in a subset of the general Italian population.
We determined α2M levels in the baseline samples of a prospective cohort (n = 19,688; age: 55 ± 12 years; 47.8 % men) of the Moli-sani study and investigated the association with the cardiovascular events (n = 432, 2.2 %) in the median follow-up period of 4.3 years. Hazard ratios (HR) with 95 % confidence intervals (CI) were calculated by multivariable Cox regression and adjusted for a large panel of confounding factors.
α2M levels above the 90th percentile were significantly associated with cardiovascular disease (CVD) events after full adjustment for age, sex, current smoking, BMI, oral contraceptive use, cardiovascular diseases, hypertension, hypercholesterolemia, diabetes and history of cancer (HR: 1.36; CI: 1.06-1.74). Moreover, high α2M was associated with coronary heart disease (CHD; HR: 1.47; CI: 1.12-1.91), but not stroke. Stratification for CVD at baseline showed that high α2M levels are associated with CHD events in subjects without CVD at baseline (HR: 1.40; CI: 1.00-1.95) and subjects with CVD at baseline (HR: 1.58; CI: 1.02-2.44).
We show in a prospective cohort that high levels of α2M could be a risk factor for cardiovascular events, especially coronary heart disease events.

Protease inhibitors of the alpha-macroglobulin family (αM) have a unique mechanism that allows them to trap proteases that is dependent not on the protease\'s class, but rather on its cleavage specificity. Proteases trigger a conformational change in the αM protein by cleaving within a \"bait region,\" resulting in the sequestering of the protease inside the αM molecule. This nonspecific inhibitory mechanism appears to have arisen early in the αM family, and the broad protease-trapping capacity that it allows may play a role in pathogen defense.Human α2-macroglobulin (A2M) is a tetrameric αM whose bait region is permissive to cleavage by most proteases, making it a broad-spectrum protease inhibitor. Recent work has demonstrated that the inhibitory capacity of A2M derives directly from its bait region sequence: modifying the bait region sequence to introduce or remove protease cleavage sites will modify A2M\'s inhibition of the relevant proteases accordingly. Thus, changing the amino acid sequence of the bait region presents an effective avenue for protein engineering of new protease inhibitors if the substrate specificity of the target protease is known. The design of new A2M-based protease inhibitors with tailored inhibitory capacities has potential applications in basic research and the clinic. In this chapter, we describe the general approach and considerations for the bait region engineering of A2M.

The universal proteinase inhibitor α2-macroglobulin (α₂-MG) exhibiting antiviral and immunomodulatory activities, is considered as an important participant in the infectious process. The activity of α₂-MG in the new coronavirus infection and post-covid syndrome (long COVID) has not been studied yet. We examined 85 patients diagnosed with community-acquired bilateral polysegmental pneumonia developed under conditions of a new coronavirus infection SARS-CoV-2. For assessment of the post-COVID period, 60 patients were examined 5.0±3.6 months after the coronavirus infection. Among these patients, 40 people had complications, manifested in the form of neurological, cardiological, gastroenterological, dermatological, bronchopulmonary symptoms. The control group included 30 conditionally healthy individuals with a negative PCR result for SARS-CoV-2 RNA and lack of antibodies to the SARS-CoV-2 virus. The α₂-MG activity in serum samples of patients with coronavirus infection dramatically decreased, up to 2.5% of the physiological level. This was accompanied by an increase in the activity of the α₁-proteinase inhibitor, elastase- and trypsin-like proteinases by 2.0-, 4.4- and 2.6-fold respectively as compared with these parameters in conditionally healthy individuals of the control. In the post-COVID period, despite the trend towards normalization of the activity of inhibitors, the activity of elastase-like and especially trypsin-like proteinases in serum remained elevated. In overweight individuals, the increase in the activity of trypsin-like proteinases was most pronounced and correlated with an increase in the antibody titer to the SARS-CoV-2 virus. In the post-COVID period, the α₂-MG activity not only normalized, but also exceeded the control level, especially in patients with dermatological and neurological symptoms. In patients with neurological symptoms or with dermatological symptoms, the α₂-MG activity was 1.3 times and 2.1 times higher than in asymptomatic persons. Low α₂-MG activity in the post-COVID period persisted in overweight individuals. The results obtained can be used to monitor the course of the post-COVID period and identify risk groups for complications.

Chandipura Virus is an emerging tropical pathogen with a high mortality rate among children. No mode of treatment or antivirals exists against CHPV infection, due to little information regarding its host interaction. Studying viral pathogen interaction with its host can not only provide valuable information regarding its propagation strategy, but also on which host proteins interact with the virus. Identifying these proteins and understanding their role in the infection process can provide more stable anti-viral targets. In this study, we focused on identifying host factors that interact with CHPV and may play a critical role in CHPV infection. We are the first to report the successful identification of Alpha-2-Macroglobulin (A2M), a secretory protein of the host that interacts with CHPV. We also established that LRP1 (Low-density lipoprotein receptor-related protein 1) and GRP78 (Glucose regulated protein 78), receptors of A2M, also interact with CHPV. Furthermore, we could also demonstrate that knocking out A2M has a severe effect on viral infection. We conclusively show the interaction of these host proteins with CHPV. Our findings also indicate that these host proteins could play a role in viral entry into the host cell.

Human α2-macroglobulin (hα2M) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hα2M undergoes stepwise transitions from its native, expanded, highly flexible, active conformation to an induced, compact, triggered conformation. As a consequence, the peptidase is entrapped by an irreversible Venus flytrap mechanism. Given the importance of hα2M, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hα2M purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hα2M is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hα2M. We therefore used a combination of native and sodium dodecylsulfate polyacrylamide gel electrophoresis, affinity and ion-exchange chromatography, multi-angle laser light scattering after size-exclusion chromatography, free cysteine quantification, and peptidase inhibition assays with endopeptidases of two catalytic classes and three protein substrates, to characterize the biochemical and biophysical properties of hα2M purified ad hoc either from fresh plasma or frozen fresh plasma after thawing. We found no differences in the molecular or functional properties of the preparations, indicating that protective components in plasma maintain native hα2M in a functionally competent state despite freezing.

Pre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with inflammatory signaling; however, the pathophysiological mechanism by which A2M is involved in PE development is not yet understood.
Human placenta samples, serum, and corresponding clinical data of the participants were collected to study the pathophysiologic mechanism underlying PE. Pregnant Sprague-Dawley rats were intravenously injected with an adenovirus vector carrying A2M via the tail vein on gestational day (GD) 8.5. Human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein endothelial cells (HUVECs), and HTR-8/SVneo cells were transfected with A2M-expressing adenovirus vectors.
In this study, we demonstrated that A2M levels were significantly increased in PE patient serum, uterine spiral arteries, and feto-placental vasculature. The A2M-overexpression rat model closely mimicked the characteristics of PE (i.e., hypertension in mid-to-late gestation, histological and ultrastructural signs of renal damage, proteinuria, and fetal growth restriction). Compared to the normal group, A2M overexpression significantly enhanced uterine artery vascular resistance and impaired uterine spiral artery remodeling in both pregnant women with early-onset PE and in pregnant rats. We found that A2M overexpression was positively associated with HUASMC proliferation and negatively correlated with cell apoptosis. In addition, the results demonstrated that transforming growth factor beta 1 (TGFβ1) signaling regulated the effects of A2M on vascular muscle cell proliferation described above. Meanwhile, A2M overexpression regressed rat placental vascularization and reduced the expression of angiogenesis-related genes. In addition, A2M overexpression reduced HUVEC migration, filopodia number/length, and tube formation. Furthermore, HIF-1α expression was positively related to A2M, and the secretion of sFLT-1 and PIGF of placental origin was closely related to PE during pregnancy or A2M overexpression in rats.
Our data showed that gestational A2M overexpression can be considered a contributing factor leading to PE, causing detective uterine spiral artery remodeling and aberrant placental vascularization.

Studies in the field of microelement speciation in the body of farm animals, in particular dairy cattle, are almost completely absent. The average concentration of Mn in the blood serum of all the studied animals (n = 80) was 2.5 μg/L, which corresponds to normal values. Of the total number of animals, 21% were the cows with the low normal values (serum Mn concentration ≤ 2 µg/L, i.e. less than Q25 of the total sample) and 25% were the animals with the high normal values (serum Mn concentration ≥ 2.72 µg/L, i.e. more than Q75 of the total sample). The data obtained in the course of the study indicate that the change in the Mn level, even in the range of normal values, is accompanied by the redistribution of this element over various protein fractions. The six found Mn blood serum forms are presumably represented by α2-macroglobulin (tetramer, dimer, and monomer), transferrin/albumine, manganese citrates, and \"free\" metal ions. The analyzed fractions of Mn found in the blood serum of cows had the following hierarchy of concentrations: in the group with low-normal values of Mn (\"free\" Mn >> tetrameric form of α2-macroglobulin >> transferrin/albumine >> dimeric form of α2-macroglobulin >> monomeric form of α2-macroglobulin >> citrate), in the group with high normal manganese values (\"free\" Mn >> monomeric form of α2-macroglobulin >> transferring/albumine >> citrate >> tetrameric form of α2-macroglobulin >> dimeric form of α2-macroglobulin). In the group with high normal Mn values relative to the group with low normal values, there was a percentage decrease in the tetrameric fraction of a2-macroglobulin from 17.2 to 4.4%, dimeric fraction of a2-macroglobulin from 6.9 to 2.2%, \"free\" Mn from 54.3 to 44.4% and an increase in monomeric fraction of a2-macroglobulin from 6.7 to 23.1%, transferrin/albumine from 10.1 to 17.7%, citrate from 4.8 to 8.2%. Our data demonstrate the features of Mn redistribution of dairy cows, which can be used for an extended assessment of the microelement status of animals.

Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.