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The Journal of Endocrinology Jun 2005Earlier studies have shown that germ cells or germ cell-conditioned media are capable of regulating alpha2-macroglobulin (alpha2-MG, a non-specific protease inhibitor)...
Earlier studies have shown that germ cells or germ cell-conditioned media are capable of regulating alpha2-macroglobulin (alpha2-MG, a non-specific protease inhibitor) expression by Sertoli cells and hepatocytes cultured in vitro. These results illustrate a possible physiological link between testes and liver regarding alpha2-MG production. Using a series of surgical procedures including castration, hemicastration, and hepatectomy coupled with Northern blot and immunoblot analyses, we report herein that the surge in alpha2-MG expression in the liver in response to inflammation is indeed regulated, at least in part, by the testis via testosterone. It was found that hepatectomy induced at least a tenfold increase in the steady-state mRNA and protein production of alpha2-MG in the liver. However, castration induced a mild but not statistically significant induction of alpha2-MG in the liver in contrast to sham operation or hemicastration alone, when hemicastration alone could induce liver alpha2-MG production by almost fourfold. Perhaps most important of all, hepatectomy accompanied by castration significantly reduced the liver alpha2-MG response to the surgery-induced inflammation compared with hepatectomy alone, illustrating that the removal of the testicles can induce a loss of signal communications between the testis and the liver, rendering a significant loss of the alpha2-MG response to experimentally induced inflammation in the liver. Interestingly, this lack of response of the liver to surgery-induced inflammation regarding alpha2-MG production following castration could be restored, at least in part, by using testosterone implants placed subdermally 6 days prior to orchiectomy. Collectively, these results illustrate that a physiological link does indeed exist between the testis and the liver, and that testes per se can influence the liver in vivo alpha2-MG expression in response to inflammation possibly via testosterone or testosterone-induced biological factor(s).
Topics: Animals; Drug Implants; Gene Expression Regulation; Hepatectomy; Immunoblotting; Inflammation; Liver; Male; Orchiectomy; RNA, Messenger; Rats; Rats, Sprague-Dawley; Surgical Wound Infection; Testis; Testosterone; alpha-Macroglobulins
PubMed: 15930176
DOI: 10.1677/joe.1.06136 -
The Journal of Biological Chemistry Aug 2022The protease inhibitor α-macroglobulin (A2M) is a member of the ancient α-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as...
The protease inhibitor α-macroglobulin (A2M) is a member of the ancient α-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as complement factor C3. A2M and other A2MF proteins undergo an extensive conformational change upon cleavage of their bait region by proteases. However, the mechanism whereby cleavage triggers the change has not yet been determined. We have previously shown that A2M remains functional after completely replacing its bait region with glycine and serine residues. Here, we use this tabula rasa bait region to investigate several hypotheses for the triggering mechanism. When tabula rasa bait regions containing disulfide loops were elongated by reducing the disulfides, we found that A2M remained in its native conformation. In addition, cleavage within a disulfide loop did not trigger the conformational change until after the disulfide was reduced, indicating that the introduction of discontinuity into the bait region is essential to the trigger. Previously, A2MF structures have shown that the C-terminal end of the bait region (a.k.a. the N-terminal region of the truncated α chain) threads through a central channel in native A2MF proteins. Bait region cleavage abolishes this plug-in-channel arrangement, as the bait region retracts from the channel and the channel itself collapses. We found that mutagenesis of conserved plug-in-channel residues disrupted the formation of native A2M. These results provide experimental evidence for a structural hypothesis in which retraction of the bait region from this channel following cleavage and the channel's subsequent collapse triggers the conformational change of A2M and other A2MF proteins.
Topics: Amino Acid Sequence; Disulfides; Protein Conformation; alpha-Macroglobulins
PubMed: 35787371
DOI: 10.1016/j.jbc.2022.102230 -
Journal of Medicine 1985
Review
Topics: Animals; Clinical Laboratory Techniques; Disease; Humans; Reference Values; alpha-Macroglobulins
PubMed: 2430038
DOI: No ID Found -
Progress in Molecular and Subcellular... 1996
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Journal of Biomolecular Structure &... Jun 2022Ifosfamide is an active alkylating chemotherapeutic drug chemically related to nitrogen mustard. The pharmacokinetics of drugs is affected upon binding with protein,...
Ifosfamide is an active alkylating chemotherapeutic drug chemically related to nitrogen mustard. The pharmacokinetics of drugs is affected upon binding with protein, making the studies on drug-protein interaction promising. The present study investigates the interaction between ifosfamide and human antiproteinase-alpha-2-macroglobulin (αM) by using multi-spectroscopic and techniques. The UV-visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods were employed to unveil the mode and mechanism of ifosfamide-αM interaction. Fluorescence quenching studies performed at three different temperatures indicated that ifosfamide-αM complex formation involves static quenching. Far UV-CD spectra revealed a minor alteration in the secondary structure of αM instigated by ifosfamide. The thermodynamic parameters determined by fluorescence quenching experiment and isothermal titration calorimetry (ITC) suggested that the complex between ifosfamide and αM involves hydrogen bonding and hydrophobic interactions. Molecular docking illustrates that ifosfamide binds with moderate affinity to Lys1240, Asn173, Ser957, Leu955, Asp953, Lys1216 and Thr1236 residues during the interaction. Molecular dynamic (MD) simulation suggested that the ifosfamide forms a stable complex with αM. Communicated by Ramaswamy H. Sarma.
Topics: Antineoplastic Agents; Binding Sites; Calorimetry; Circular Dichroism; Female; Humans; Ifosfamide; Molecular Docking Simulation; Pregnancy; Pregnancy-Associated alpha 2-Macroglobulins; Protein Binding; Spectrometry, Fluorescence; Thermodynamics
PubMed: 33267704
DOI: 10.1080/07391102.2020.1852115 -
Reproductive Biology and Endocrinology... Oct 2011Alpha 2 macroglobulin (A2M; also known as ovostatin), a homotetrameric protein with four disulfide-linked subunits, has the unique feature of inactivating/inhibiting...
BACKGROUND
Alpha 2 macroglobulin (A2M; also known as ovostatin), a homotetrameric protein with four disulfide-linked subunits, has the unique feature of inactivating/inhibiting most known proteases including serine-, threonine-, cysteine-, aspartic- and metalloproteases. In chickens, A2M has been identified and characterized biochemically, but little is known of its functional role(s) in the oviduct, hormonal regulation of expression or its expression in ovarian carcinomas in chickens. Therefore, we investigated estrogen regulation of A2M gene expression during development of the chicken oviduct, and its expression in normal and cancerous ovaries from chickens.
METHODS
To determine tissue-specific expression of A2M in chickens, we collected various organs from male and female chickens and performed RT-PCR analyses. To examine A2M gene expression in the oviduct of 1-week-old female chicks that received a subcutaneous implant of 15 mg DES in the abdominal region for 20 days, we performed RT-PCR, qPCR and in situ hybridization analyses using cDNAs from control- (n=5) and DES-treated oviducts (n=5), and then each segment of the oviduct from DES-treated chicks. To determine if A2M is a biomarker of ovarian cancer in hens, we collected cancerous (n=10) ovaries from a total of 136 chickens which had completely stopped egg-laying and performed RT-PCR and in situ hybridization analyses.
RESULTS
We found that A2M is most abundant in the chicken oviduct, specifically luminal (LE) and glandular epithelia (GE), but it was not detected in any other tissues of either sex. We then determined that DES (dietylstilbestrol, a synthetic nonsteroidal estrogen) increased A2M mRNA only in LE and GE of the oviduct of chicks. Further, expression of A2M was most abundant in GE of endometrioid adenocarcinoma of cancerous, but not normal ovaries of hens.
CONCLUSIONS
Collectively, results of the present study indicate that A2M is novel estrogen-stimulated gene expressed in LE and GE of the chicken oviduct and may be used for monitoring effects of therapies for ovarian cancer in laying hens.
Topics: Animals; Avian Proteins; Biomarkers, Tumor; Carcinoma; Chickens; Diethylstilbestrol; Epithelial Cells; Estrogens, Non-Steroidal; Female; Gene Expression Regulation, Developmental; Macroglobulins; Male; Neoplasm Proteins; Organ Specificity; Ovarian Neoplasms; Ovary; Oviducts; Phylogeny; Poultry Diseases; RNA, Messenger; Sequence Homology, Amino Acid; alpha-Macroglobulins
PubMed: 21978460
DOI: 10.1186/1477-7827-9-137 -
Journal of Biochemistry Oct 1993alpha-Macroglobulin and murinoglobulin were purified to homogeneity from Syrian hamster plasma and their properties were compared with those of their respective homologs... (Comparative Study)
Comparative Study
alpha-Macroglobulin and murinoglobulin were purified to homogeneity from Syrian hamster plasma and their properties were compared with those of their respective homologs from other mammals. The trypsin-inhibiting capacity of hamster murinoglobulin was much weaker than those of rat and mouse murinoglobulins. Hamster alpha-macroglobulin was cleaved by trypsin at a number of sites whereas the human homolog was split essentially only in a "bait" region into two fragments of similar size. Hamster alpha-macroglobulin treated with methylamine differed from that treated with trypsin in the electrophoretic mobility, intensity of fluorescence induced by binding of bis(8-anilino-1-naphthalenesulfonate), and plasma clearance pattern, whereas virtually no difference was observed between the human homologs treated in the same manner. The reaction of hamster alpha-macroglobulin with methylamine, as measured by the generation of thiol groups and the decrease in trypsin-protein amidase activity, was much slower than that of the human homolog. Trypsin in a complex with hamster alpha-macroglobulin retained its fibrinolytic activity, but this was not the case for human or rabbit alpha-2-macroglobulin. These results suggest that, compared with the human homolog, hamster alpha-macroglobulin is more loosely packed in the native state, undergoes conformational change more slowly on treatment with methylamine, and less efficiently hinders the access of proteinaceous substrates to trapped proteinase. The serum concentration of hamster alpha-macroglobulin was 6.9 mg/ml, or about 3-fold higher than that of the human type, and showed little change during the acute-phase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Amidohydrolases; Animals; Cricetinae; Electrophoresis, Polyacrylamide Gel; Female; Fibrinolysis; Hot Temperature; Humans; Male; Mesocricetus; Methylamines; Mice; Molecular Weight; Pancreatic Elastase; Serum Globulins; Spectrometry, Fluorescence; Sulfhydryl Compounds; Trypsin; Trypsin Inhibitors; alpha-Macroglobulins
PubMed: 7506251
DOI: 10.1093/oxfordjournals.jbchem.a124209 -
The Journal of Clinical Endocrinology... Mar 1995alpha 2-Macroglobulin (A2M) is a broad spectrum plasma protease inhibitor previously described in uterine effluents and recently demonstrated to bind and possibly...
alpha 2-Macroglobulin (A2M) is a broad spectrum plasma protease inhibitor previously described in uterine effluents and recently demonstrated to bind and possibly modulate the functions of cytokines. As cytokines, proteases, and protease inhibitors are important in implantation and endometrial physiology, we sought to investigate and characterize the pattern of production of A2M in the endometrium. Endometrial tissues from different phases of the menstrual cycle were analyzed for A2M production. Tissues were incubated in methionine-free Minimum Essential Medium with [35S]methionine for 12 h at 37 C in 5% CO2. Conditioned media were immunoprecipitated with a polyclonal antibody to human A2M. Recovered proteins were resolved under reducing and nonreducing conditions by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by autoradiography. Immunohistochemistry was performed on both cryostat sections of OCT-embedded tissue and formalin-fixed paraffin-embedded tissue. The intensity of staining was evaluated, and a histochemical score was assigned. Comparisons between histochemical scores were performed using the nonparametric Wilcoxon rank sum test. Immunoprecipitation with A2M antibody yielded a single 320-kilodalton protein band under nonreducing conditions and a single 182-kilodalton band under reducing conditions. Both physical and immunological properties of the recovered protein were consistent with A2M. A2M was identified in all endometrial samples and represented approximately 2% of the total radiolabeled proteins produced. Immunohistochemical analysis revealed prominent stromal, but no glandular, staining for A2M throughout the menstrual cycle. The stromal staining in secretory endometrial samples was significantly more intense than that in proliferative samples. In the proliferative phase, staining was more intense in the spongiosum and basalis layers, and less intense in the superficial compactum layer. In the secretory phase, it remained very intense in the spongiosum, but less so in the basalis layer. These findings indicate that A2M is predominantly produced by the stromal component of endometrial tissue. This production is menstrual cycle dependent, with zonal differences in A2M expression within the endometrium, which may indicate a functional significance relevant to the process of implantation that merits further investigation.
Topics: Endometrium; Female; Humans; Immunohistochemistry; Precipitin Tests; alpha-Macroglobulins
PubMed: 7533769
DOI: 10.1210/jcem.80.3.7533769 -
Nihon Ketsueki Gakkai Zasshi : Journal... Aug 1962
Topics: Globulins; Humans; Macroglobulins; Serum Globulins
PubMed: 13954746
DOI: No ID Found -
Klinicheskaia Laboratornaia Diagnostika Jun 2000