Mechanisms of proteolytic activity regulation exerted via a unique propeptide in matrix metalloproteinases and intra/intermolecular interactions in a novel family of minimal gluzincins

Abstract: Metallopeptidases are major players in the physiology and pathology of all living organisms. Their exquisite regulation is therefore essential for proper function and to prevent misdirected temporal and/or spatial proteolytic activity, which may lead to disease. This regulation is achieved through a wide variety of mechanisms, including their biosynthesis as inactive precursors, also known as zymogens. In the present thesis, various mechanisms of metallopeptidase regulation were studied by using a combination of biochemical, biophysical, and structural techniques. In the first project, the crystal structure of a zymogen fragment of Tannerella forsythia karilysin revealed the shortest propeptide reported to date for a metallopeptidase. Additional biochemical and biophysical assays allowed ascertaining the importance of the propeptide in protein expression, folding and stability. In the second project, a novel family of minimal prokaryotic metallopeptidases termed ? minigluzincins? was discovered, providing a minimal soluble scaffold for gluzincin metallopeptidases and integral-membrane metallopeptidases. Two members of this family, called proabylysin and projannalysin, showed two unique zymogenic forms of latency maintenance exerted, respectively, via intramolecular and intermolecular interactions through their C-terminal segments. In the third project, a further member of this novel family, called selecase, evinced selective and specific proteolytic activity against casein. A set of biophysical and structural studies showed that selecase reversibly commutes between several conformations of defined three-dimensional structure, which are associated with loss of enzymatic activity due to autoinhibition (i.e. active monomers vs. inactive dimers, tetramers and octamers). Overall, the present thesis contributes substantially to the field broadening previous knowledge at the molecular level on metallopeptidases and their regulatory mechanisms, which paves the way for the design of specific inhibitors to modulate their activity as part of therapeutic approaches. Resum en Catala: Les metal·lopeptidases participen de manera decisiva en la fisiologia i patologia de tots els organismes vius. La seva estricta regulacio es, per tant, essencial pel correcte funcionament d? aquestes i per a prevenir una activitat proteolitica inadequada en el temps i/o espai que podria causar malalties. Aquesta regulacio s? aconsegueix mitjancant un ampli ventall de mecanismes, incloent la seva biosintesis com a precursors inactius, tambe coneguts com a zimogens. En la present tesi s? han estudiat diversos mecanismes de regulacio de metal·lopeptidases, tot combinant tecniques bioquimiques, biofisiques i estructurals. En el primer projecte, l? estructura cristal·lina d? un fragment zimogenic de la proteina ? karilysin? de Tannerella forsythia ha revelat el propeptid mes curt descrit fins al moment per una metal·lopeptidasa. Assajos bioquimics i biofisics addicionals han permes determinar la importancia del propeptid en l? expressio, plegament i estabilitat de la proteina. En el segon projecte, s? ha descobert una nova familia de metal·lopeptidases minimes d? origen procariota, que s? han anomenat ? minigluzincins? , les quals proporcionen un model estructural per a metal·lopeptidases pertanyents al clan de les gluzincines i per a d? altres integrals de membrana. Dues membres d? aquesta familia, anomenades ? proabylysin? i ? projannalysin? , han exhibit formes uniques de manteniment de latencia exercides, respectivament, de forma intra- i intermolecular mitjancant llurs segments C-terminals. En el tercer projecte, una altra membre d? aquesta nova familia, anomenada ? selecase? , ha presentat una activitat proteolitica selectiva i especifica sobre caseina. La conjuncio d? estudis duts a terme, tant biofisics com estructurals, ha evidenciat que ? selecase? transita de manera reversible entre diverses conformacions d? estructura tridimensional definida, associades amb una disminucio d? activitat enzimatica deguda a autoinhibicio (es a dir, monomers actius i dimers, tetramers i octamers inactius). En definitiva, aquesta tesi contribueix de manera substancial a ampliar el coneixement previ sobre metal·lopeptidases a nivell molecular i a entendre els mecanismes que en regulen l? activitat, facilitant aixi el disseny d? inhibidors especifics com a part d? aproximacions terapeutiques.