kDa Fragment Research Articles

Granzyme B cleaves Tenascin-C to release its C-terminal domain in rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune disorders and is characterized by exacerbated joint inflammation that can lead to tissue remodeling and autoantigen generation. Despite the well-documented accumulation of the serine protease Granzyme B (GzmB) in the biospecimens of patients with RA, little is understood pertaining to its role in pathobiology. In the present study Tenascin-C (TN-C), a large extracellular matrix glycoprotein and an endogenous trigger of inflammation, was identified as a substrate for GzmB in RA. GzmB cleaves TN-C in vitro to generate three fragments: a 130 kDa fragment that remains anchored to the matrix, and two 70 and 30 kDa fragments that are released and solubilized. Mass spectrometry results seem to indicate that the 30 kDa fragment generated by GzmB most likely contains TN-C pro-inflammatory C-terminal fibrinogen-like domain. Soluble levels of GzmB and TN-C are also significantly elevated in the synovial fluids of RA patients compared to healthy controls, with two 70 kDa and 30 kDa soluble TN-C fragments detectable in the synovial fluids of RA patients. The molecular weights of these fragments coincide with those generated by GzmB in vitro, suggesting that GzmB also cleaves TN-C in RA patients. Granzyme K (GzmK), another member of the granzyme family, also cleaves TN-C in vitro. However, unlike GzmB, the molecular weights of TN-C fragments generated by GzmK in vitro do not correspond to fragments identified in patients. Altogether, our data supports the contribution of Granzyme B, but not Granzyme K, to RA through the cleavage of Tenascin-C.

PDGF-induced internalisation promotes proteolytic cleavage of PDGFRβ in mesenchymal cells

Platelet-derived growth factor (PDGF)-induced signalling via PDGF receptor β (PDGFRβ) leads to activation of downstream signalling pathways which regulate multiple cellular responses. It is unclear how PDGFRβ is degraded; both lysosomal and proteasomal degradation have been suggested. In this study, we have characterised the proteolytic cleavage of ligand-activated PDGFRβ, which results in two fragments: a larger fragment containing the extracellular domain, the transmembrane segment, and a part of the intracellular juxtamembrane region with a molecular mass of ∼130 kDa, and an intracellular ∼70 kDa fragment released into the cytoplasm. The proteolytic processing did not take place without internalisation of PDGFRβ. In addition, chelation of intracellular Ca2+ inhibited proteolytic processing. Inhibition of the proteasome affected signal transduction by increasing the phosphorylation of PDGFRβ, PLCγ, and STAT3 while reducing it on Erk1/2 and not affecting Akt. The proteolytic cleavage was observed in fibroblasts or cells that had undergone epithelial-mesenchymal transition.

Targeting Cleavage of C-Terminal Fragment of Cytoskeletal Filamin A in Cancers.

Human cancers express altered levels of actin-binding cytoskeletal filamin A (FLNA) protein. FLNA in mammals consists of an actin-binding domain at its N-terminus that is followed by 24 immunoglobulin-like repeat modules interrupted by two hinge regions between repeats 15-16 and 23-24. Cleavage of these hinge regions produces a naturally occurring C-terminal 90 kDa fragment of FLNA (FLNACT) that physically interacts with multiple proteins with diverse functions. This cleavage leads to actin cytoskeleton remodeling, which in turn contributes to cellular signaling, nucleocytoplasmic shuttling of transcriptional factors and nuclear receptors, and regulation of their transcriptional activities that are important for initiation and progression of cancers. Therefore, recent studies have proposed blocking FLNA cleavage as a means of cancer therapy. Here, we update how FLNA cleavage has been targeted by different approaches and their potential implications for future treatment of human cancers.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

Recombinant human enamelin produced in Escherichia coli promotes mineralization in vitro

BackgroundEnamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent.ResultsIn the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml.ConclusionsThese findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.

Differences in Cardiac Troponin T Composition in Myocardial Infarction and End-Stage Renal Disease Patients: A Blood Tube Effect?

Cardiac troponin T (cTnT) is key in diagnosing myocardial infarction (MI) but is also elevated in end-stage renal disease (ESRD) patients. Specific larger cTnT proteoforms were identified for the acute phase of MI, while in serum of ESRD patients solely small cTnT fragments were found. However, others allocated this to a pre-analytic effect due to abundant thrombin generation in serum. Therefore, we investigated the effect of various anticoagulation methods on cTnT composition and concentration and compared the cTnT composition of MI and ESRD patients. The agreement of cTnT concentrations between simultaneously collected serum, lithium-heparin (LH) plasma, and ethylenediaminetetraacetic acid (EDTA) plasma was studied using the high-sensitivity (hs-)cTnT immunoassay. cTnT proteoform composition was investigated in a standardized time-dependent manner through spike experiments and in simultaneously collected blood matrixes of MI and ESRD patients. Excellent hs-cTnT concentration agreements were observed across all blood matrixes (slopes > 0.98; 95% CI, 0.96-1.04). Time-dependent degradation (40 kDa intact:29 kDa fragment:15 to 18 kDa fragments) was found in LH plasma and EDTA plasma, and serum in ratios (%) of 90:10:0, 0:5:95, and 0:0:100, respectively (48 h after blood collection). Moreover, gel filtration chromatography (GFC) profiles illustrated mainly larger cTnT proteoforms in MI patients, while in ESRD patients mainly 15 to 18 kDa fragments were found for all matrices. The extent of cTnT degradation in vitro is dependent on the (anti)coagulation method, without impacting hs-cTnT concentrations. Furthermore, mainly larger cTnT proteoforms were present in MI patients, while in ESRD patients mainly small 15 to 18 kDa cTnT fragments were found. These insights are essential when developing a novel hs-cTnT assay targeting larger cTnT proteoforms.

Mice Mutated in the First Fibronectin Domain of Adhesion Molecule L1 Show Brain Malformations and Behavioral Abnormalities.

The X-chromosome-linked cell adhesion molecule L1 (L1CAM), a glycoprotein mainly expressed by neurons in the central and peripheral nervous systems, has been implicated in many neural processes, including neuronal migration and survival, neuritogenesis, synapse formation, synaptic plasticity and regeneration. L1 consists of extracellular, transmembrane and cytoplasmic domains. Proteolytic cleavage of L1's extracellular and transmembrane domains by different proteases generates several L1 fragments with different functions. We found that myelin basic protein (MBP) cleaves L1's extracellular domain, leading to enhanced neuritogenesis and neuronal survival in vitro. To investigate in vivo the importance of the MBP-generated 70 kDa fragment (L1-70), we generated mice with an arginine to alanine substitution at position 687 (L1/687), thereby disrupting L1's MBP cleavage site and obliterating L1-70. Young adult L1/687 males showed normal anxiety and circadian rhythm activities but enhanced locomotion, while females showed altered social interactions. Older L1/687 males were impaired in motor coordination. Furthermore, L1/687 male and female mice had a larger hippocampus, with more neurons in the dentate gyrus and more proliferating cells in the subgranular layer, while the thickness of the corpus callosum and the size of lateral ventricles were normal. In summary, subtle mutant morphological changes result in subtle behavioral changes.

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58 kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58 kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.

Abstract 1549: Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer

Abstract Background: CD146, an adhesion molecule involved in inflammatory regulation, is linked to increased aggressiveness in Triple Negative Breast Cancer (TNBC). Yet, the molecular mechanisms of CD1146-driven TNBC metastasis in an inflammatory context remain elusive. We synthesized CD146 nanobody (anti-CD146 #112-2), which degraded CD146 protein specifically, inhibiting IL-6-mediated JAK/STAT3 activation and suppressing TNBC metastasis. This study aims to elucidate the mechanisms of CD146 degradation by 112-2 and the role of CD146 in mediating inflammatory responses leading to TNBC metastasis. Methods: CD146 knockout and overxpression cell models were created using CRISPR/Cas9 or lentiviral techniques. Lung metastasis was evaluated by injecting luciferase-labeled cells into the tail veins of Balb/c-nu mice, monitored with the IVIS Kinetic system. The impact of CD146 on TNBC metastasis in an inflammatory environment was explored through cell-adhesion assays and trans-endothelial migration assays using CD146 knockout models and HUVEC cells. Transwell assays assessed the influence of IL-6 and 112-2 on TNBC. CD146 mutant vectors with specific truncations were generated to confirm the binding sites between CD146 and 112-2. ELISA and immunofluorescence assays examined CD146 protein internalization. CD146 degradation by 112-2 was confirmed using proteasome inhibitor MG132 and lysosome inhibitor Bafilomycin A1, along with western blotting and immunofluorescence experiments. Results: Stimulation of TNBC cells with IL-6 increased CD146 expression and tumor metastasis. Knocking down CD146 significantly reduced IL-6 secretion by TNBC cells, leading to reduced tumor-endothelial cell adhesion and trans-endothelial migration. CD146 knockout or 112-2 treatment inhibited IL-6-mediated JAK/STAT3 activation and tumor metastasis. Experiments with CD146 mutants and molecular docking identified the specific 112-2 binding site at leucine positions 331-332 of CD146 protein. 112-2 treatment led to CD146 protein internalization and cleavage, resulting in an 80 kDa fragment recognized by both 112-2 and C-terminus-specific CD146 antibodies, ultimately inhibiting the activation of IL-6/JAK/STAT3 signaling. Notably, the inhibition of proteasome or lysosome activity failed to reverse the 112-2-induced reduction in CD146 protein levels. However, inhibiting lysosomal acidification resulted in significant co-localization of CD146 with lysosomes, suggesting a potential role of lysosomes in 112-2-mediated cleavage of CD146. Conclusion: CD146 promotes TNBC metastasis by modulating IL-6/JAK/STAT3 activation. The nanobody anti-CD146 #112-2 specifically binds to CD146 extracellular domain induces the proteolysis of CD146 protein, thereby inhibiting the tumor-promoting effect of IL-6. Citation Format: Kaiyuan Huang, Hexing Sun, Chengyu Wu, Danping Lin, Jinling Chen, Yanhan Chen, Xinqiang Fang, Shuzhao Chen, Yuanke Liang, Haoyu Lin. Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1549.

The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

Legumain is a lysosomal cysteine protease that has been implicated in an increasing amount of physiological and pathophysiological processes. However, the upstream mechanisms regulating the expression and function of legumain are not well understood. Here, we provide in vitro and in vivo data showing that vitamin D3 (VD3) enhances legumain expression and function. In turn, legumain alters VD3 bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP). Active VD3 (1,25(OH)2D3) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Upregulation of legumain was also observed in vivo, evidenced by increased legumain mRNA in the liver and spleen, as well as increased legumain activity in kidneys from wild-type mice treated with 25(OH)D3 (50 µg/kg, subcutaneously) for 8 days compared to a control. In addition, the serum level of legumain was also increased. We further showed that active legumain cleaved purified VDBP (55 kDa) in vitro, forming a 45 kDa fragment. In vivo, no VDBP cleavage was found in kidneys or liver from legumain-deficient mice (Lgmn-/-), whereas VDBP was cleaved in wild-type control mice (Lgmn+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D3 and total VD3 and altered expression of key renal enzymes involved in VD3 metabolism (CYP24A1 and CYP27B1). In conclusion, a regulatory interplay between VD3 and legumain is suggested.

Filamin A is involved in human intrahepatic cholangiocarcinoma aggressiveness and progression.

Intrahepatic cholangiocarcinoma (iCCA) is a primary liver tumour, characterized by poor prognosis and lack of effective therapy. The cytoskeleton protein Filamin A (FLNA) is involved in cancer progression and metastasis, including primary liver cancer. FLNA is cleaved by calpain, producing a 90 kDa fragment (FLNACT ) that can translocate to the nucleus and inhibit gene transcription. We herein aim to define the role of FLNA and its cleavage in iCCA carcinogenesis. We evaluated the expression and localization of FLNA and FLNACT in liver samples from iCCA patients (n = 82) revealing that FLNA expression was independently correlated with disease-free survival. Primary tumour cells isolated from resected iCCA patients expressed both FLNA and FLNACT , and bulk RNA sequencing revealed a significant enrichment of cell proliferation and cell motility pathways in iCCAs with high FLNA expression. Further, we defined the impact of FLNA and FLNACT on the proliferation and migration of primary iCCA cells (n = 3) and HuCCT1 cell line using silencing and Calpeptin, a calpain inhibitor. We observed that FLNA silencing decreased cell proliferation and migration and Calpeptin was able to reduce FLNACT expression in both the HuCCT1 and iCCA cells (p < .05 vs. control). Moreover, Calpeptin 100 μM decreased HuCCT1 and primary iCCA cell proliferation (p <.00001 vs. control) and migration (p < .05 vs. control). These findings demonstrate that FLNA is involved in human iCCA progression and calpeptin strongly decreased FLNACT expression, reducing cell proliferation and migration.

Blood-Based Diagnostic Assay for λ Light Chain Amyloidosis through Quantification of an Amyloidogenicity-Indicating Neo-Epitope

Light chain amyloidosis (AL) is a rare, progressive, and often fatal protein misfolding disease. AL is caused by aberrant clonal plasma cell proliferation, where excess amounts of amyloidogenic monoclonal Free Light Chain (FLC) are produced, secreted into the circulation, misfolded and deposted as amyloid fibrils in various vital organs, such as the heart, kidney, liver, and nerves. The involed lambda (λ) FLC accounts for ~75% of all AL cases. Amyloidogenic FLCs (aFLCs) are believed to be the toxic species contributing to AL disease pathology. Specific quantification of aFLCs can provide critical guidance in clinical practice but has been hampered by the LCs sequence diversity as well as the lack of tests that are able to differentiate between circulating normal polyclonal FLCs and aFLCs. Contrary to non-amyloidogenic, aFLCs are kinetically and oftentimes thermodynamically unstable. aFLCs are prone to misfolding and formation of pathogenic conformations such as non-native structures, proteolytic fragments, soluble aggregates, and amyloid fibrils. We exploited such characteristics of aFLCs by applying limited proteolysis using Proteinase K, where less stable λ aFLC proteins readily yield a 23KDa fragment composed of the homodimeric LC constant domain (dLCCD) linked by a disulfide bond. Formation of the dLCCD fragment reveals a highly conserved, novel, cryptic epitope at the N-terminus of the fragment. We have generated monoclonal antibodies (mAbs) that explicitly bind to such novel epitope and developed immunoassays for the specific detection and sensitive quantification of λ aFLCs in biological samples. Western blot or Meso Scale Discovery (MSD) analyses were employed to detect and quantify dLCCD signal using the dLCCD mAbs on plasma samples from randomly selected, treatment naïve and λ-restricted cases with biopsy-proven AL amyloidosis before and after exposure to optimized proteolytic conditions. Tissue amyloid deposits of λ-type were confirmed in most AL cases. Informed consent for data and plasma sample collections were obtained from the Institutional Review Boards and in accordance with the Declaration of Helsinki. Multiple myeloma (MM) plasma samples of unknown LC restriction type, treatment or amyloid deposits status and normal volunteer control samples were purchased from BioIVT (USA). A set of representative results based on three runs are shown in Fig. 1. As little as 2 µL of biofluid was used for each assay. Upon limited proteolysis, dLCCD signal was detected in 32 of 35 AL plasmas, including two samples from patients with AL-associated IgM lymphoplasmacytic lymphoma (LPL). Of the three dLCCD negative AL samples, one presented with monoclonal IgAλ, and two cases were bi-clonal on serum immunofixation electrophoresis. dLCCD signal was not detected in 18 of 19 MM plasma samples. Similarly, dLCCD signal was undetectable in 23 normal plasmas. Overall, the dLCCD signals detected in AL plasmas were 10x-200x above that of normal control and most MM samples (Fig. 1). The low limit of quantification of the MSD assay was ~0.4 ng/dL (1.4 pM), with &amp;gt;500x dynamic range, enabling sensitive and robust quantification of λ aFLCs in biosamples. Analysis of Proteinase K recognition potential of the cleavage site of 389 λ LC sequences from the AL-Base, a database of amyloidogenic LC sequences (https://wwwapp.bumc.bu.edu/BEDAC_ALBase/) predicted that the dLCCD assay can detect λ aFLCs in up to 99% of λ AL patients, irrespective of the FLC sequences. This was confirmed with the 100% detection rate of the dLCCD signal in a validation cohort of 35 additional monoclonal λ-restricted AL samples. In summary, we have developed a quantitative blood-based immunoassay that is sensitive and highly specific for λ aFLC irrespective of the protein sequences. The preliminary data demonstrates the potential of our assay for AL diagnostics. Additional clinical applications for future exploration will include (i) monitoring and early detection of AL amyloidosis in plasma cell dyscrasias (monoclonal gammopathy of unknown significance, MM, and smoldering MM) and lymphoproliferative disorders, including LPL; and (ii) clinical management of patients with AL amyloidosis via the assessment and monitoring of minimal residual disease and therapeutic responses. The utility of the dLCCD assay in the quantification of λ aFLCs in bi-clonal AL cases warrants further investigation.

Role of prophenoloxidase 1 from the beetle Octodonta nipae in melanized encapsulation of a wasp egg

Exploring the function of the host immune system can help to understand the host-parasitoid interaction. Prophenoloxidase (PPO) is crucial in defensive melanization during the encapsulation of wasp eggs. However, the existence of multiple PPO sequences increases the difficulty of exploring the specific functions of individual PPOs. We previously identified three PPOs in the nipa palm hispid beetle, Octodonta nipae. Our current work showed that OnPPO1 and OnPPO2 possessed the typical characteristics of the type III copper family, but OnPPO3 lacked the conserved histidine residues, and its active sites were substituted with Gln. OnPPOs showed the highest expression in hemocytes, but OnPPO3 presented extremely low abundance compared with that of OnPPO1 and OnPPO2, and only OnPPO1 showed a quick response after wasp infection. OnPPO1 knockdown decreased the encapsulation index and inhibited melanization, whereas silencing of OnPPO3 appeared to have no adverse effect on encapsulation and melanization, and silencing of OnPPO2 presented low RNAi efficiency. Moreover, the cleavage of recombinant OnPPO1 produced a 62 kDa fragment with high PO activity. OnPPO1 could be produced by oenocytoids, granulocytes and plasmatocytes, and was distributed around wasp eggs during capsule formation. Overall, our results indicate that proteolytic cleavage of OnPPO1 plays key roles in the melanized encapsulation of wasp eggs. This finding illuminates the mechanism of PPO activation in this invasive beetle and provides guidance for its biological control.

Soluble E-cadherin participates in BLM-induced pulmonary fibrosis by promoting EMT and lung fibroblast migration.

Soluble E-cadherin (sE-cad) is an 80 kDa fragment derived from E-cadherin that is shed from the cell surface through proteolytic cleavage and is a biomarker in various cancers that promotes invasion and migration. Alveolar epithelial destruction, aberrant lung fibroblast migration and inflammation contribute to pulmonary fibrosis. Here, we hypothesized that E-cadherin plays an important role in lung fibrosis. In this study, we found that E-cadherin was markedly increased in the bronchoalveolar lavage fluid (BALF) and serum of mice with pulmonary fibrosis and that blocking sE-cad with HECD-1, a neutralizing antibody targeting the ectodomain of E-cadherin, effectively inhibited myofibroblast accumulation and collagen deposition in the lungs after bleomycin (BLM) exposure. Moreover, transforming growth factor-β (TGF-β1) induced the shedding of sE-cad from A549 cells, and treatment with HECD-1 inhibited epithelial-mesenchymal transition (EMT) stimulated by TGF-β1. Fc-E-cadherin (Fc-Ecad), which is an exogenous form of sE-cad, robustly promoted lung fibroblast migration. E-cadherin participates in bleomycin (BLM)-induced lung fibrosis by promoting EMT in the alveolar epithelium and fibroblast activation. E-cadherin may be a novel therapeutic target for lung fibrosis.

Effects of truncations in the N- and C-terminal domains of filensin on filament formation with phakinin in cell-free conditions and cultured cells.

Filensin and phakinin are lens fiber cell-specific proteins that constitute the beaded filaments that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N- and C-terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30-416) and Fil(30-369), respectively, were examined. Fil(30-416) lacks the N-terminal 29 amino acids and the C-terminal 248 amino acids. Fil(30-369) lacks the N-terminal 29 residues and the C-terminal 295 residues. In cell-free assembly characterized by electron microscopy, filensin and Fil(30-416) co-polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30-369) formed only aggregates. In cultured SW-13 and MCF-7 cells expressing fluorescent fusion proteins, filensin and Fil(30-416) co-polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30-369) gave aggregates. Therefore, while truncation of the N-terminal 29 amino acids did not affect filament formation, truncation of the C-terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370-416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with beaded filament formation and thereby contribute to opacification.

Molecular Mechanisms of Endothelialitis in SARS-CoV-2 Infection: Evidence for VE-Cadherin Cleavage by ACE2.

Long COVID-19 syndrome appears after Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection with acute damage to microcapillaries, microthrombi, and endothelialitis. However, the mechanisms involved in these processes remain to be elucidated. All blood vessels are lined with a monolayer of endothelial cells called vascular endothelium, which provides a the major function is to prevent coagulation. A component of endothelial cell junctions is VE-cadherin, which is responsible for maintaining the integrity of the vessels through homophilic interactions of its Ca++-dependent adhesive extracellular domain. Here we provide the first evidence that VE-cadherin is a target in vitro for ACE2 cleavage because its extracellular domain (hrVE-ED) contains two amino acid sequences for ACE2 substrate recognition at the positions 256P-F257 and 321PMKP-325L. Indeed, incubation of hrVE-ED with the active ectopeptidase hrACE2 for 16 hrs in the presence of 10 μM ZnCl2 showed a dose-dependent (from 0.2 ng/μL to 2 ng/μL) decrease of the VE-cadherin immunoreactive band. In vivo, in the blood from patients having severe COVID-19 we detected a circulating form of ACE2 with an apparent molecular mass of 70 kDa, which was barely detectable in patients with mild COVID-19. Of importance, in the patients with severe COVID-19 disease, the presence of three soluble fragments of VE-cadherin (70, 62, 54 kDa) were detected using the antiEC1 antibody while only the 54 kDa fragment was present in patients with mild disease. Altogether, these data clearly support a role for ACE2 to cleave VE-cadherin, which leads to potential biomarkers of SARS-CoV-2 infection related with the vascular disease in "Long COVID-19".

In-depth characterization of myofibrillar muscle proteome changes in lambs fed hazelnut skin by-products

This study investigated the effect of hazelnut skin by-products supplementation on lamb myofibrillar proteome changes during post-mortem storage (0, 4, and 7 days). Gel-based proteomics and bioinformatics approaches were applied to reveal the underlying biochemical pathways and their importance in lamb meat texture development. Twenty-two Valle del Belice male lambs were randomly assigned to two dietary treatments: control (C) for lambs fed with maize-barley diet, and hazelnut skin (H) for lambs fed hazelnut skin by-product as maize partial replacer in the concentrate diet (150 g/kg DM basis). A greater myofibrillar fragmentation index (MFI) was found in meat from the C group at day 0 of storage (91.33 vs 98.38 in H and C groups, respectively). Conversely, starting from 4 days of storage, higher MFI values were observed in meat from lambs fed hazelnut skin (113.74 and 116.1 vs 99.28 and 107.26 in H and C groups at 4 and 7 days, respectively). Myofibrillar proteome changes estimated by SDS-PAGE and immunoblotting revealed a degradation of desmin (P<0.01) and troponin T (P<0.001) intact proteins, and an increase in the abundance of the appearing 30 kDa fragment (P<0.001) after 4 days of storage in meat from H than the C group. In-depth proteomics and bioinformatics revealed 44 proteoforms (26 unique proteins), mainly involved in actin filament-based process/cytoskeleton organization, energy metabolism, and heat shock proteins, as the major interconnected pathways impacted by hazelnut by-product feeding strategy on lamb meat quality. Twelve proteins were proposed in this trial as potential biomarkers of lamb meat texture as a consequence of hazelnut skin by-products supplementation.

Peripаrtal Cardiomyopathy

The purpose of the study is to describe a clinical case of peripartal cardiomyopathy (CM) and conduct literature analysis. Peripartal CM is defined as reduced ejection fraction of the left ventricle &lt;45%, reduced fractional shortening &lt;30%, as well as increased end-diastolic size of the left ventricle &gt; 2.7 cm/m2. At the same time, there should be no detectable causes of heart failure, as well as development of heart failure in the last month of pregnancy or within 5 months after childbirth. The review presents data on possible causes of peripartal CM. Risk factors for peripartal CM are: age younger than 20 years and older than 30 years, preeclampsia, hypertension, multiple pregnancy, prolonged duration of viral infection, macro- and micronutrient insufficiency, etc. Prolactin and its 16 kDa fragment, as well as excessive activity of cathepsin D, play a leading role in pathogenesis of peripartal CM. A step-by-step algorithm for diagnosis of peripartal CM in real clinical conditions is described. Material and methods. The patient is K., 18 years old, in anamnesis: no prior history of cardiovascular diseases, according to the patient, she had pneumonia a year ago. The patient has not registered increased blood pressure. Physiological pregnancy proceeded satisfactorily; natural delivery was on time. On the 56th day after childbirth, the patient was admitted to the intensive care unit with complaints: shortness of breath, muscle weakness, palpitations, lower leg edema. From anamnesis: 1 month after childbirth, the next day after psychoemotional overstrain, the patient began to notice shortness of breath, lack of sleep, muscle weakness, decreased lactation. The examination revealed an increase in the sedimentation rate of erythrocytes, C-reactive blood protein, proteinuria, hematuria, as well as a decrease in the ejection fraction of the left ventricle. Results. Together with standard therapy, addition of prolactin secretion inhibitors and dopamine receptor stimulators led to significant improvement of the patient’s condition: proteinuria significantly decreased, edematous syndrome was gone, and LV contractile function significantly recovered. Conclusion. The clinical case demonstrates the development of a rare variant of peripartal CM, debuting with proteinuria and preserved kidney function, as well as normal serum prolactin levels. Apparently, there are different phenotypes of peripartal CM.

Apoptotic Cell Death in Bicuspid-Aortic-Valve-Associated Aortopathy.

The bicuspid aortic valve (BAV) is the most common cardiovascular congenital abnormality and is frequently associated with proximal aortopathy. We analyzed the tissues of patients with bicuspid and tricuspid aortic valve (TAV) regarding the protein expression of the receptor for advanced glycation products (RAGE) and its ligands, the advanced glycation end products (AGE), as well as the S100 calcium-binding protein A6 (S100A6). Since S100A6 overexpression attenuates cardiomyocyte apoptosis, we investigated the diverse pathways of apoptosis and autophagic cell death in the human ascending aortic specimen of 57 and 49 patients with BAV and TAV morphology, respectively, to identify differences and explanations for the higher risk of patients with BAV for severe cardiovascular diseases. We found significantly increased levels of RAGE, AGE and S100A6 in the aortic tissue of bicuspid patients which may promote apoptosis via the upregulation of caspase-3 activity. Although increased caspase-3 activity was not detected in BAV patients, increased protein expression of the 48 kDa fragment of vimentin was detected. mTOR as a downstream protein of Akt was significantly higher in patients with BAV, whereas Bcl-2 was increased in patients with TAV, assuming a better protection against apoptosis. The autophagy-related proteins p62 and ERK1/2 were increased in patients with BAV, assuming that cells in bicuspid tissue are more likely to undergo apoptotic cell death leading to changes in the wall and finally to aortopathies. We provide first-hand evidence of increased apoptotic cell death in the aortic tissue of BAV patients which may thus provide an explanation for the increased risk of structural aortic wall deficiency possibly underlying aortic aneurysm formation or acute dissection.

Resveratrol improves episodic-like memory and motor coordination through modulating neuroinflammation in old rats

Strategies focus on the use of molecules found in food derived from plants as polyphenols, arouse interest for combating aging. The polyphenol resveratrol, due to its antioxidants and anti-inflammatory properties, is increasingly studied, being still unknown their effects on episodic-like memory and motor coordination. This research evaluated the in vivo effect of chronic resveratrol treatment (20 mg/kg/day, i.p, 28 days) in episodic-like memory and motor coordination in non-pathological old rats, comparing with young and old vehicle-treated rats, using the novel object recognition and the rotarod tests. In combination with the analyses of the effect of resveratrol on the immunoreactivity of the main molecular proteins involved in neuroinflammation (SIRT1, SIRT1-75 kDa fragment, NF-κB acetylation) in the hippocampus of these rats. Results suggest a beneficial effect of resveratrol on episodic-like memory and in motor coordination through modulating neuroinflammation, acting on SIRT1 and NF-κB signaling pathway in the hippocampus of old rats.