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International Journal of Molecular... Mar 2023Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated...
Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated protein based on the three-dimensional structure of the Z-domain derived from Staphylococcal protein A. This HED retains µM binding activity to the human IgG1CH2-CH3 elbow region. We determined the crystal structure of HED in association with IgG1's Fc. This demonstrated that HED preserves the same three-bundle helix structure and Fc-interacting residues as the Z domain. HED was fused to the single chain variable fragment (scFv) of mAb 4D5 to produce an antibody-like protein capable of interacting with the p185Her2/neu ectodomain and the Fc of IgG. When further fused with murine IFN-γ (mIFN-γ) at the carboxy terminus, the novel species exhibited antitumor efficacy in vivo in a mouse model of human breast cancer. The HED is a novel platform for the therapeutic utilization of engineered proteins to alleviate human disease.
Topics: Humans; Animals; Mice; Female; Single-Chain Antibodies; Breast Neoplasms; Staphylococcal Protein A
PubMed: 37047449
DOI: 10.3390/ijms24076477 -
Brain, Behavior, and Immunity Jul 2023Low-grade inflammation may occur in association with several mental disorders of early adulthood, though associations with markers of chronic inflammation such as...
Associations between plasma inflammatory markers and psychotic disorder, depressive disorder and generalised anxiety disorder in early adulthood: A nested case-control study.
BACKGROUND
Low-grade inflammation may occur in association with several mental disorders of early adulthood, though associations with markers of chronic inflammation such as soluble urokinase plasminogen activator receptor (suPAR) are less well-established. We aimed to examine associations between acute and chronic inflammatory markers and mental disorders, as well as psychiatric co-morbidity, in young adults aged 24 years in the Avon Longitudinal Study of Parents and Children.
METHODS
Included were 781 participants (of 4019 who attended at age 24 years) who completed psychiatric assessments and provided plasma samples. Of these, 377 met criteria for psychotic disorder, depressive disorder or generalised anxiety disorder and 404 did not. Plasma concentrations of IFN-γ, IL-6, IL-8, IL-10, TNF-α, CRP, sVCAM1, sICAM1, suPAR and alpha-2-macroglobulin were measured using immunoassays. Logistic regression compared standardised inflammatory marker levels in cases and controls. Negative binomial regression evaluated associations between inflammatory markers and co-morbidity (number of mental disorders). Models were adjusted for sex, body mass index, cigarette smoking, cannabis use and employment status, then additionally for childhood trauma.
RESULTS
For psychotic disorder, there was evidence for associations with IL-6 (odds ratio[OR] 1.68, 95 %CI 1.20-2.34) and suPAR (OR 1.74, 95 %CI 1.17-2.58). There was weaker evidence for an association between suPAR and depressive disorder (OR 1.31, 95 %CI 1.05-1.62). There was little evidence for associations between inflammatory markers and generalised anxiety disorder. There was weak evidence for an association between suPAR and co-morbidity (β 0.10, 95 %CI 0.01-0.19). There was little evidence for additional confounding by childhood trauma.
CONCLUSIONS
There was evidence that 24-year-olds with psychotic disorder had raised plasma IL-6 and suPAR concentrations compared to controls. These findings have implications regarding the role of inflammation in mental disorders in early adulthood.
Topics: Child; Young Adult; Humans; Adult; Receptors, Urokinase Plasminogen Activator; Biomarkers; Longitudinal Studies; Case-Control Studies; Interleukin-6; Psychotic Disorders; Inflammation; Anxiety Disorders; Depressive Disorder
PubMed: 37004760
DOI: 10.1016/j.bbi.2023.03.025 -
Journal of Thrombosis and Haemostasis :... Jul 2023ADAMTS13 is a circulating metalloprotease that cleaves von Willebrand factor (VWF) in a shear-dependent manner. ADAMTS13 is secreted as an active protease but has a long...
BACKGROUND
ADAMTS13 is a circulating metalloprotease that cleaves von Willebrand factor (VWF) in a shear-dependent manner. ADAMTS13 is secreted as an active protease but has a long half-life, suggesting that it is resistant to circulating protease inhibitors. These zymogen-like properties indicate that ADAMTS13 exists as a latent protease that is activated by its substrate.
OBJECTIVES
To investigate the mechanism of ADAMTS13 latency and resistance to metalloprotease inhibitors.
METHODS
Probe the active site of ADAMTS13 and variants using alpha-2 macroglobulin (A2M), tissue inhibitors of metalloproteases (TIMPs), and Marimastat.
RESULTS
ADAMTS13 and C-terminal deletion mutants are not inhibited by A2M, TIMPs, or Marimastat, but cleave FRETS-VWF73, suggesting that the metalloprotease domain is latent in the absence of substrate. Within the metalloprotease domain, mutating the gatekeeper triad (R193, D217, D252) or substituting the calcium-binding (R180-R193) or the variable (G236-S263) loops with corresponding features from ADAMTS5 did not sensitize MDTCS to inhibition. However, substituting the calcium-binding loop and an extended variable loop (G236-S263) corresponding to the S1-S1' pockets with those from ADAMTS5, resulted in MDTCS-GVC5 inhibition by Marimastat, but not by A2M or TIMP3. Substituting the MD domains of ADAMTS5 into full-length ADAMTS13 resulted in a 50-fold reduction in activity compared with the substitution into MDTCS. However, both chimeras were susceptible to inhibition, suggesting that the closed conformation does not contribute to the latency of the metalloprotease domain.
CONCLUSION
The metalloprotease domain protects ADAMTS13 from inhibitors and exists in a latent state that is partially maintained by loops flanking the S1 and S1' specificity pockets.
Topics: Humans; von Willebrand Factor; ADAM Proteins; Calcium; Hydroxamic Acids; ADAMTS13 Protein
PubMed: 36990157
DOI: 10.1016/j.jtha.2023.03.021 -
Journal of Medical Biochemistry Mar 2023To clarify if a2-macroglobulin (α2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD).
BACKGROUND
To clarify if a2-macroglobulin (α2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD).
METHODS
The content of α2M and reactive oxygen species (ROS) were measured to compare mildly and severely degenerated human nucleus pulposus (NP) tissue by immunohistochemistry, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Additionally, exogenic α2M was used to culture severely degenerated NP tissue in vitro. The effects of α2M on hypochlorite (HOCl)-treated NP cells were evaluated, containing antioxidative enzymes, ROS level, collagen II, and aggrecan expression, MMP3/13, and ADAMTS4/5.
RESULTS
ROS level increased in severely degenerated NP, accompanying with a decreased α2M content. Supplement of α2M could decrease the ROS level of cultured NP in vitro, meanwhile, the MMP13 and ADAMTS4 expression were also reduced. It was found that treatment of HOCl resulted in oxidative damage to NP cells and decreased α2M expression in a dose and time-dependent manner. Furthermore, exogenic α2M stimulation reversed the HOCl-triggered ROS accumulation. The promotion of SOD1/2, CAT, GPX1, collagen II, and aggrecan, and suppression of MMP3/13, ADAMTS4/5 expression caused by α2M were also observed.
CONCLUSIONS
Our study indicates that α2M has an antioxidative ability in degenerated NP cells by promoting the antioxidative enzyme production.
PubMed: 36987418
DOI: 10.5937/jomb0-39557 -
Life (Basel, Switzerland) Mar 2023Intense exercise can cause inflammation and oxidative stress due to the production of reactive oxygen species. These pathophysiological processes are interdependent, and...
Dietary Supplementation with Boswellia serrata, Verbascum thapsus, and Curcuma longa in Show Jumping Horses: Effects on Serum Proteome, Antioxidant Status, and Anti-Inflammatory Gene Expression.
Intense exercise can cause inflammation and oxidative stress due to the production of reactive oxygen species. These pathophysiological processes are interdependent, and each one can induce the other, creating a vicious circle. A placebo-controlled blind study was carried out in show jumping horses (n. 16) to evaluate the effects of a commercial dietary supplement (Dolhorse N.B.F. Lanes srl, Milan, Italy) containing Verbascum thapsus leaf powder (1.42%), Curcuma longa (14.280 mg/kg), and Boswellia serrata (Roxb ex Colebr) (14.280 mg/kg) extracts. Before and after 10 days of dietary supplementation, blood samples were collected to evaluate the protein levels, antioxidants, and inflammatory responses by proteomic analysis or real-time Reverse Transcriptase-Polymerase Chain Reaction (real-time RT-PCR). A total of 36 protein spots, connected to 29 proteins, were modulated by dietary supplementation, whereas real-time RT-PCR revealed a significant downregulation of proinflammatory cytokines (interleukin 1α ( < 0.05) and interleukin-6 (0.005), toll-like receptor 4 ( < 0.05), and IKBKB ( < 0.05) in supplemented sport horses. Immunoglobulin chains, gelsolin, plasminogen, vitamin D binding protein, apolipoprotein AIV, and filamin B were overexpressed, whereas haptoglobin, α-2-HS-glycoprotein, α2-macroglobulin, afamin, amine oxidase, 60S acidic ribosomal protein, and complement fragments 3, 4, and 7 were reduced. No effect was observed on the antioxidant defense systems. The present results suggest this phytotherapy may reinforce the innate immune responses, thus representing a valid adjuvant to alleviate inflammation, which is a pathophysiological process in sport horses.
PubMed: 36983904
DOI: 10.3390/life13030750 -
Scientific Reports Mar 2023Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many...
Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many age-related diseases. Within the cell, molecular chaperones facilitate the refolding of misfolded proteins into their bioactive forms, thus preventing undesirable interactions and aggregation. Although the mechanisms of intracellular protein degradation pathways for intracellular misfolded proteins have been extensively studied, the protein degradation pathway for extracellular proteins remain poorly understood. In this study, we identified several misfolded proteins that are substrates for alpha 2-macroglobulin (αM), an extracellular chaperone. We also established a lysosomal internalization assay for αM, which revealed that αM mediates the lysosomal degradation of extracellular misfolded proteins. Comparative analyses of αM and clusterin, another extracellular chaperone, indicated that αM preferentially targets aggregation-prone proteins. Thus, we present the degradation pathway of α2M, which interacts with aggregation-prone proteins for lysosomal degradation via selective internalization.
Topics: Female; Pregnancy; Humans; Pregnancy-Associated alpha 2-Macroglobulins; Protein Folding; Proteostasis; Proteolysis; Transcription Factors; Lysosomes
PubMed: 36977730
DOI: 10.1038/s41598-023-31104-x -
The Journal of Maternal-fetal &... Dec 2023Spontaneous preterm birth (delivery before 37 completed weeks) is the single most important cause of perinatal morbidity and mortality. The rate is increasing world-wide...
Spontaneous preterm birth (delivery before 37 completed weeks) is the single most important cause of perinatal morbidity and mortality. The rate is increasing world-wide with a great disparity between low, middle and high income countries. It has been estimated that the cost of neonatal care for preterm babies is more than 4 times that of a term neonate admitted into the neonatal care. Furthermore, there are high costs associated with long-term morbidity in those who survive the neonatal period. Interventions to stop delivery once preterm labor starts are largely ineffective hence the best approach to reducing the rate and consequences is prevention. This is either primary (reducing or minimizing factors associated with preterm birth prior to and during pregnancy) or secondary - identification and amelioration (if possible) of factors in pregnancy that are associated with preterm labor. In the first category are optimizing maternal weight, promoting healthy nutrition, smoking cessation, birth spacing, avoidance of adolescent pregnancies and screening for and controlling various medical disorders as well as infections prior to pregnancy. Strategies in pregnancy, include early booking for prenatal care, screening and managing medical disorders and their complications, and identifying predisposing factors to preterm labor such as shortening of the cervix and timely instituting progesterone prophylaxis or cervical cerclage where appropriate. The use of biomarkers such as oncofetal fibronectin, placental alpha-macroglobulin-1 and IGFBP-1 where cervical screening is not available or to diagnosis PPROM would identify those that require close monitoring and allow the institution of antibiotics especially where infection is considered a predisposing factor. Irrespective of the approach to prevention, timing the administration of corticosteroids and where necessary tocolysis and magnesium sulfate are associated with an improved outcome. The role of genetics, infections and probiotics and how these emerging dimensions help in the diagnosis of preterm birth and consequently prevention are exciting and hopefully may identify sub-populations for targeted strategies.
Topics: Adolescent; Female; Humans; Infant, Newborn; Pregnancy; Early Detection of Cancer; Obstetric Labor, Premature; Placenta; Premature Birth; Uterine Cervical Neoplasms
PubMed: 36966809
DOI: 10.1080/14767058.2023.2183756 -
Pakistan Journal of Medical Sciences 2023To evaluate the clinical efficacy of continuous veno-venous hemofiltration (CVVH) combined with hemoperfusion for the treatment of multiple myeloma (MM) complicated with...
OBJECTIVE
To evaluate the clinical efficacy of continuous veno-venous hemofiltration (CVVH) combined with hemoperfusion for the treatment of multiple myeloma (MM) complicated with acute kidney injury (AKI).
METHODS
Medical records of 73 patients with MM complicated with AKI admitted to the First People's Hospital of Huzhou from January 2019 to January 2021 were retrospectively analyzed. According to the treatment records, 35 patients received simple chemotherapy (control group), and 38 patients received CVVH combined with HP on the basis of chemotherapy (observation group). We compared the clinical efficacies, renal function indexes, and the serum globulin and erythrocyte sedimentation rate (ESR) values between the two groups.
RESULTS
After the treatment, the total efficacy of the observation group was significantly higher (81.58%) than that in the control group (57.14%; <0.05). Serum cystatin C (CysC), urea nitrogen (BUN), β macroglobulin (β-MG) and creatinine (SCr) levels were significantly lower in the observation group than in the control group ( <0.05). Serum globulin level and ESR values in the observation group after the treatment were also significantly lower than in the control group ( <0.05).
CONCLUSIONS
The outcomes of patients with MM complicated with AKI treated with CVVH and hemoperfusion differ significantly from those of the patients treated only with CVVH. Combining CVVH and hemoperfusion helps to improve the efficacy of the treatment, promotes renal function recovery, and improves the levels of serum globulin and ESR.
PubMed: 36950391
DOI: 10.12669/pjms.39.2.6966 -
Journal of Cellular Biochemistry May 2023Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a...
Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α -macroglobulin (α M) and tissue-type plasminogen activator (tPA). In macrophages, α M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.
Topics: Humans; Cytokines; Membrane Proteins; Plasminogen Inactivators; Diabetes Mellitus, Type 2; Endoplasmic Reticulum Chaperone BiP; N-Methylaspartate; Macrophages; Antibodies; Low Density Lipoprotein Receptor-Related Protein-1
PubMed: 36947703
DOI: 10.1002/jcb.30401 -
Scientific Reports Mar 2023Human α-macroglobulin (hαM) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hαM...
Human α-macroglobulin (hαM) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hαM 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 hαM, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hαM purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hαM is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hαM. 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 hαM 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 hαM in a functionally competent state despite freezing.
Topics: Humans; Endopeptidases; Freezing; Macroglobulins; Peptide Hydrolases; Plasma
PubMed: 36941303
DOI: 10.1038/s41598-023-31800-8