The recognition of collagen and triple-helical toolkit peptides by MMP-13: sequence specificity for binding and cleavage.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.This work was supported by a British Heart Foundation programme grant, RG/009/003/27122, and peptide synthesis, by grants from Medical Research Council and Wellcome Trust.This is the author accepted manuscript. The final version can be found on the publisher's website at: http://www.jbc.org/content/early/2014/07/09/jbc.M114.58344

MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1

Matrix metalloproteinase 13 (MMP-13) degrades collagenous extracellular matrix and its aberrant activity associates with diseases such as arthritis, cancer, atherosclerosis and fibrosis. The wide range of MMP-13 proteolytic capacity suggests that it is a powerful, potentially destructive proteinase and thus it has been believed that MMP-13 is not produced in most adult human tissues in the steady state. Present study has revealed that human chondrocytes isolated from healthy adults constitutively express and secrete MMP-13, but that it is rapidly endocytosed and degraded by chondrocytes. Both pro- and activated MMP-13 bind to clusters II and III of low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). Domain deletion studies indicated that the hemopexin domain is responsible for this interaction. Binding competition between MMP-13 and ADAMTS-4, -5 or TIMP-3, which also bind to cluster II, further shown that the MMP-13 binding site within cluster II is different from those of ADAMTS-4, -5 or TIMP-3. MMP-13 is therefore co-endocytosed with ADAMTS-5 and TIMP-3 by human chondrocytes. These findings indicate that MMP-13 may play a role on physiological turnover of cartilage extracellular matrix and that LRP1 is a key modulator of extracellular levels of MMP-13 and its internalization is independent of the levels of ADAMTS-4, -5 and TIMP-3

将教育理解为: 种具有拯救特性的关怀

يتطلب هذا العدد الخاص المتعلق بالتعليم في عصرنا هذا التفكير في ما يغذي التعليم, وما يحافظ عليه معًا: الرعاية.على الرغم من أن التفكير في التعليم والرعاية ليس بالأمر الجديد, إلا أنه اكتسب اهتمامًا متجددًا في الجائحة, عندما كان الفصل بين الطلاب والمعلمين غير واضح بسبب التجربة الجماعية التي كنا نعيشها. في وقت سابق, منذ أوائل التسعينيات, كان عمل Nel Noddings أساسيًا لأولئك الذين أرادوا إعادة التأكيد على أبعاد الرفاهية في التعليم. قام هذا المؤلف بتحليل الرعاية ومكانتها في الأخلاق, ووضع رؤية لأهمية الرعاية في التعليم والتعلم بشكل عام. منذ ذلك الحين, استمر المتخصصون في أخلاقيات الرعاية في تطوير مفاهيم حول ماهية الرعاية الجيدة, وتوجيههم نحو السياقات المؤسسية والاجتماعية التي تعزز مجتمعات ومجتمعات الرعاية. يعتمد هذا العمل على هذه الدراسة. هو لقاء بين مفاهيم Robert Stake حول التعليم والمقاربات المستقبلة للتقييم وخبرات Merel Visse في مجال أخلاقيات الرعاية الصحية, والتي تُرجمت لجمهور عام من الأشخاص الذين ليسوا بالضرورة من علماء الأخلاق. أدى هذا التفاعل إلى ظهور كتاب A Paradigm of Care, وفي إسبانيا, إلى كتيب يحتوي على خمسة عشر جانبًا من جوانب الرعاية التي نشاركها مع أعضاء هيئة التدريس وطلاب الدراسات العليا في المدرسة الصيفية المذكورة. تهدف هذه الجوانب إلى تحفيز التفكير والتداول والاستماع, والتي تعتبر مهمة جدًا للرعاية.This Special Issue on Education in the Present Age needs a reflection on what nurtures education, holds it together: care. This essay is an account of our experiences with care in education. It is based on our contribution to the International Doctoral Summer School in Granada in June 2022.  Thinking about education and care is not new, but gained renewed attention in the pandemic, when the lines between students and teachers blurred because of the collective experience we were going through. Before that, since the early 199o’s, Nel Noddings’ work is key for those wanting to reaffirm the caring dimensions of education. She analyzed care and its place in ethics, and developed a view on the importance of care in schooling and learning in general. Since then, care ethicists further developed notions of what is good care, turning those to institutional and societal contexts that promote caring communities and societies. Our essay builds on that work. It is an encounter between Robert Stake’s notions of education and responsive approaches to evaluation and Merel Visse’s experiences with the field of care ethics, translated for a general audience of people who are not necessarily ethicists. Our encounter resulted in the book A Paradigm of Care, and in Spain, resulted in a leaflet with fourteen facets of care we shared with the graduate faculty and students. These insights are meant to evoke and stimulate reflection, deliberation and listening: important to care.Este número especial sobre la educación en la época actual requiere una reflexión sobre lo que alimenta la educación, lo que la mantiene unida: el cuidado. Este trabajo es un relato de algunas experiencias con la atención educativa.  Aunque pensar en educación y cuidados no es nuevo, cobró renovada atención en la pandemia, cuando la separación entre alumnos y profesores se difuminaron por la experiencia colectiva que estábamos viviendo. Desde principios de los 90, el trabajo de Nel Noddings fue clave para quienes querían reafirmar las dimensiones asistenciales de la educación. Esta autora analizó el cuidado y su lugar en la ética, y desarrolló una visión sobre la importancia del cuidado en la escolarización y el aprendizaje. Desde entonces, los especialistas en la ética del cuidado han seguido desarrollando nociones de lo que es un buen cuidado, orientándolas hacia contextos institucionales y sociales que promueven comunidades y sociedades solidarias. Este trabajo se basa en ese estudio. Es un encuentro entre las nociones de Robert Stake sobre la educación y los enfoques receptivos de la evaluación y las experiencias de Merel Visse en el campo de la ética asistencial, traducido para un público general de personas que no son necesariamente especialistas en ética. Esta interacción dio lugar al libro A Paradigm of Care y, en España, a un folleto con catorce aspectos de los cuidados que compartimos con el profesorado y los estudiantes de posgrado. Estos aspectos pretenden estimular la reflexión, la deliberación y la escucha tan importantes para el cuidado.Este número especial sobre a educação na época atual exige uma reflexão sobre o que alimenta a educação, o que a mantém unida: o cuidado. Este artigo é um relato de algumas experiências com os cuidados educativos.  Embora pensar em educação e cuidados não seja novo, ganhou uma atenção renovada na pandemia, quando a separação entre alunos e professores se esbateu pela experiência coletiva que estávamos a viver. A partir do início dos anos 1990, o trabalho de Nel Noddings foi fundamental para quem queria reafirmar as dimensões assistenciais da educação. Esta autora analisou o cuidado e o seu lugar na ética, e desenvolveu uma visão sobre a importância do cuidado na escolaridade e na aprendizagem. Desde então, os especialistas na ética do cuidado têm continuado a desenvolver noções do que é um bom cuidado, orientando-as para contextos institucionais e sociais que promovem comunidades e sociedades solidárias. Este trabalho baseia-se nesse estudo. Trata-se de um encontro entre as noções de Robert Stake sobre a educação e as abordagens recetivas da avaliação e as experiências de Merel Visse no domínio da ética assistencial, traduzido para um público geral de pessoas que não são necessariamente especialistas em ética. Esta interação deu origem ao livro “A Paradigm of Care” e, em Espanha, a uma brochura com quatorze aspetos dos cuidados que partilhamos com os professores e os estudantes de pós-graduação nesta escola de Verão. Estes aspetos destinam-se a estimular a reflexão, a deliberação e a escuta, tão importantes para o cuidado.Este número especial sobre a educação na época atual exige uma reflexão sobre o que alimenta a educação, o que a mantém unida: o cuidado. Este artigo é um relato de algumas experiências com os cuidados educativos.  Embora pensar em educação e cuidados não seja novo, ganhou uma atenção renovada na pandemia, quando a separação entre alunos e professores se esbateu pela experiência coletiva que estávamos a viver. A partir do início dos anos 1990, o trabalho de Nel Noddings foi fundamental para quem queria reafirmar as dimensões assistenciais da educação. Esta autora analisou o cuidado e o seu lugar na ética, e desenvolveu uma visão sobre a importância do cuidado na escolaridade e na aprendizagem. Desde então, os especialistas na ética do cuidado têm continuado a desenvolver noções do que é um bom cuidado, orientando-as para contextos institucionais e sociais que promovem comunidades e sociedades solidárias. Este trabalho baseia-se nesse estudo. Trata-se de um encontro entre as noções de Robert Stake sobre a educação e as abordagens recetivas da avaliação e as experiências de Merel Visse no domínio da ética assistencial, traduzido para um público geral de pessoas que não são necessariamente especialistas em ética. Esta interação deu origem ao livro “A Paradigm of Care” e, em Espanha, a uma brochura com quatorze aspetos dos cuidados que partilhamos com os professores e os estudantes de pós-graduação nesta escola de Verão. Estes aspetos destinam-se a estimular a reflexão, a deliberação e a escuta, tão importantes para o cuidado.这是一篇对当代教育进行反思的特殊文章，该文探讨了“关怀”如何滋养教育，维系教育这一整体。尽管对教育及关怀的思考早已不是新奇的主题，但是在疫情期间，在这个特殊的集体经历让学生及教师间的分隔变得模糊不清的时期，它又重新吸引了人们的注意力。在九十年代初期，内尔·诺丁斯的研究就在重新肯定教育支援范畴上起到了非常关键的作用。诺丁斯分析了关怀及其在伦理方面的角色，为理解关怀在学校教育和学习中的重要性延展出了崭新的视角。在此基础上，更多的关怀伦理学专家继续研究，发展出了更多的关于良好关怀的概念，并将其应用到社会和机构的背景下，以此推动团结坚定的社区及社会关系。该文章以此进行研究，将罗伯特·斯塔克的回应式教育评估概念与梅蕾尔·维塞尔在伦理支援方面的经验融合，展现给更广泛的不一定拥有伦理学专业知识的普通大众。在两种理论的互动中诞生了众多成果，如《关怀典范》这本著作和在西班牙发表的宣传册。这本关于关怀的十五个方面的宣传册是上述国际博士暑期学校教师和学生共同分享使用的。通过宣传册中涉及的多个方面，旨在鼓励更多的思考和倾听，因为对关怀来说，这些都是至关重要的内容

Interaction between triple-helical collagens and human collagenases

Collagens are the major structural proteins in animal tissues. Their degradation is essential in embryogenesis and development, while unbalanced collagen breakdown is seen in diseases such as arthritis, atherosclerosis and cancer. Fibril-­forming collagens I, II and III consist of three polypeptide chains forming a triple helix, which is resistant to cleavage by most proteases. Collagenases of the matrix metalloproteinase family (MMP-­1, MMP-­8, and MMP-­13) degrade fibrillar collagens by locally unwinding the helix, followed by cleavage into ¼ and ¾ fragments. They comprise two domains, the catalytic (Cat) domain and the hemopexin (Hpx) domain, which are connected via a flexible linker. Both domains are essential for collagenolysis, but the exact sites of collagenase-­collagen interactions and how they unwind collagen remain elusive. This thesis addresses the roles of individual collagenase domains, and the sites in both the enzyme and the substrate that are involved in collagen binding and unwinding, focusing on the fibril-­forming collagens and human MMP-­1 as a prototype. MMP-­1 bound to immobilised collagen I with markedly higher affinity than its Hpx domain alone. The Cat domain alone failed to bind to collagen, but in the full-­length enzyme it participated in collagen binding. Above 25°C the two-­ domain binding involved the catalytic site cleft. Triple-­helical peptide (THP) Toolkits of collagens II and III were screened for MMP-­1 binding, and the collagenase binding motif has been established. It contains two hydrophobic residues within a 9 residue distance. Finally, hydrogen/deuterium exchange mass spectrometry (H/DXMS) experiments indicated two potential collagen binding sites: 285-­316 and 349-365 in the Hpx domain, and suggested a possibility of a dynamic interaction of the collagenase N-­terminus with collagen. These results imply that the two domains of collagenase bind to collagen in a cooperative manner. Based on the THP binding and H/DXMS data a 3D model of collagenase-­collagen interaction has been proposed. It assumes that collagenase utilises hydrophobic interactions to unwind the collagen helix via perturbation of the hydrogen-­bond network which stabilises the helix.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

STAT3 precedes HIF1α transcriptional responses to oxygen and oxygen and glucose deprivation in human brain pericytes

Brain pericytes are important to maintain vascular integrity of the neurovascular unit under both physiological and ischemic conditions. Ischemic stroke is known to induce an inflammatory and hypoxic response due to the lack of oxygen and glucose in the brain tissue. How this early response to ischemia is molecularly regulated in pericytes is largely unknown and may be of importance for future therapeutic targets. Here we evaluate the transcriptional responses in in vitro cultured human brain pericytes after oxygen and/or glucose deprivation. Hypoxia has been widely known to stabilise the transcription factor hypoxia inducible factor 1-alpha (HIF1α) and mediate the induction of hypoxic transcriptional programs after ischemia. However, we find that the transcription factors Jun Proto-Oncogene (c-JUN), Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells (NFκB) and signal transducer and activator of transcription 3 (STAT3) bind genes regulated after 2hours (hs) of omitted glucose and oxygen before HIF1α. Potent HIF1α responses require 6hs of hypoxia to substantiate transcriptional regulation comparable to either c-JUN or STAT3. Phosphorylated STAT3 protein is at its highest after 5 min of oxygen and glucose (OGD) deprivation, whereas maximum HIF1α stabilisation requires 120 min. We show that STAT3 regulates angiogenic and metabolic pathways before HIF1α, suggesting that HIF1α is not the initiating trans-acting factor in the response of pericytes to ischemia