Biology subjects

Moali, C.

Publications and source records attributed to Moali, C..

7 recordsLinked to original sources

bioRxiv2026

A recombinant heterotrimeric mini-procollagen I reveals new functional and structural roles for the α2(I) chain

Collagen I provides mechanical stability to organs and plays a critical role in several physiological processes such as wound healing and bone remodeling. Its ability to form fibrils depends on the proteolytic conversion of soluble procollagen I into mature protomers. However, the mechanistic aspects of this critical step have been difficult to analyze due to the lack of recombinant procollagen I suitable for structural and biochemical analyses. Here, we describe the successful production and purification of a heterotrimeric mini-procollagen I [1(I)22(I)] together with its homotrimeric counterpart [1(I)3] in HEK293-F cells. These tools were used to probe the specific role of the 2(I) chain both in procollagen I assembly and proteolytic maturation. While the presence of 2(I) in the heterotrimer does not strengthen trimer interfaces on its own, as revealed by cryo-electron microscopy, it provides a clear functional advantage to the procollagen substrate that is cleaved more efficiently by bone morphogenetic protein 1 (BMP-1) than the homotrimer. Furthermore, we demonstrate that the first cleavage event in the heterotrimer is at the level of the 2(I) chain, through a mechanism involving its C-telopeptide and a specific PRS motif close to the cleavage site. The preferential cleavage of 2(I) by BMP-1 can be partially corrected in the presence of PCPE-1 (procollagen C-proteinase enhancer 1) but the efficacy of the correction depends on its position on the procollagen trimer. Finally, we solved the high-resolution structure of the complex between the heterotrimer and PCPE-1 and proposed a new mechanism to explain the enhancing activity of PCPE-1.

biochemistry↗

bioRxiv2026

Structure of PCPE-2 in complex with the BMP-1 metalloprotease reveals the molecular basis of its mechanism of inhibition

PCPE-2 (procollagen C-proteinase enhancer-2) is an extracellular glycoprotein playing dual functions in the regulation of BMP-1/tolloid-like proteinases. Like its homologue PCPE-1, PCPE-2 can enhance the proteolytic maturation of fibrillar procollagens, but it also acts as a potent and specific inhibitor of BMP-1 through formation of a high-affinity complex with the protease. Here, we investigated the molecular basis of this inhibitory interaction using complementary biochemical, biophysical and structural approaches. We show that the two CUB domains of PCPE-2 must be covalently linked for efficient BMP-1 binding and inhibition, consistent with cooperative engagement of the protease. Cryo-electron microscopy combined with density-guided structural modelling and hydrogen/deuterium-exchange mass spectrometry supports a bipartite interaction complex in which the CUB1 domain of PCPE-2 engages the catalytic domain of BMP-1, while the CUB2 domains of both proteins interact together. The interaction involves the calcium-binding surfaces of the PCPE-2 CUB domains that also participate in procollagen recognition, providing a structural framework for understanding the mutually exclusive interactions of PCPE-2 with BMP-1 and procollagen. Allosteric inhibition is supported by the binding of PCPE-2 CUB1 opposite to the active site cleft, leading to the shielding of the residues surrounding the S1-prime pocket, as observed by HDX-MS. Together, these findings define the architecture of the inhibitory PCPE-2/BMP-1 complex and provide the molecular basis for understanding the distinct regulatory activities of the two procollagen C-proteinase enhancers.

biochemistry↗

bioRxiv2026

Distinct regulation of collagen biosynthesis by hypoxia and vitamin C uncovers an essential role for CXCL12-CXCR4 signaling

Collagen biosynthesis is a complex multistep process, essential for functional tissue formation and repair. It is highly active during embryogenesis and wound healing, leading to physiological fibrogenesis, while its dysregulation induces pathologies affecting connective tissues, namely fibrosis. There are several key cellular microenvironmental factors regulating collagen biosynthesis, including vitamin C, oxygen tension and growth factors. Whilst the function of these distinct factors has previously been studied individually, how they operate together, synergistically or not, is not fully characterized. Using primary human dermal fibroblasts, we demonstrated that ascorbic acid (AA) and hypoxia both induced collagen I deposition when applied individually and had additive effects when combined together. Their effects were associated to the expected increase in collagen prolyl hydroxylation. However, AA induced a much stronger collagen secretion compared to hypoxia, while hypoxia leads to a higher collagen I hydroxylation. This observation challenged the common view of collagen prolyl hydroxylation being the central mechanism of AA-induced collagen secretion. Using proteomic analysis of the secretome, biochemical and cell imaging approaches, we further showed that increased CXCL12 extracellular levels and activation of its cognate CXCR4 receptor are at the core of AA-induced collagen intracellular trafficking and secretion. This mechanism is specific to AA and is not involved under hypoxia. The existence of distinct mechanisms underlying the effects of AA and hypoxia is further demonstrated by the different ultrastructure of collagen fibrils in the two conditions. Together, our results contribute to explain how these two triggers drive collagen biosynthesis and pave the way for potential pro- or anti-fibrotic strategies aimed at improving tissue repair.

cell biology↗

bioRxiv2024

Kinetic properties of optogenetic DNA editing by LiCre-loxP

Advances in optogenetics now allow to specifically modify the DNA of live cells with light. However, using these technologies successfully requires to know their properties in terms of sensitivity, efficiency, kinetics and mechanism. We previously developed an optogenetic tool made of a single chimeric protein called LiCre that enables the induction of specific changes in the genome with blue light via DNA recombination between loxP sites [1]. Here, we used in vitro and in vivo experiments combined with kinetic modeling to provide a deeper characterization of the photo-activated LiCre-loxP recombination reaction. We find that LiCre binds DNA with high affinity in absence of light stimulus and that this binding is cooperative although not as much as for the Cre recombinase from which LiCre was derived. In yeast, addition of riboflavin to the culture medium had no effect on LiCres efficiency, even when cells over-expressed riboflavin kinase, suggesting that abundance of the flavin mono-nucleotide co-factor is not limiting for the reaction. However, LiCres efficiency in yeast gradually increased when raising temperature from 20{degrees}C to 37{degrees}C. The recombination kinetics observed in live cells are best explained by a model where photo-activation of two or more DNA-bound LiCre units (happening in seconds) can produce (in several minutes) a functional recombination synapse. This model was able to capture the effect of a point mutation altering LiCres light cycle. This deeper understanding of the LiCre-loxP system provides additional knowledge for designing experiments where specific genetic changes are induced in live cells with light. AUTHOR SUMMARYUsing a technology called optogenetics, scientists are now able to change the DNA sequence of live cells by illuminating them with light. In theory, they can trigger in genetically-engineered organisms a mutation of interest in specific cells at a specific time. This practice, however, is not common because optogenetics relies on light-controlled enzymes that are recent and not well characterized. We previously developed one of these enzymes, called LiCre, which recognizes a specific piece of DNA. Following illumination with blue light, LiCre can switch on or off whatever gene is close by. Here, we combined experiments and computational modeling to better understand how, and how fast, LiCre works. We find that, although it is inactive in the dark, LiCre does not need photo-activation to bind to its DNA partner. We estimated the speed at which LiCre gets deactivated in the dark and the speed at which active LiCre molecules modify DNA. We also showed the effect of temperature and illumination dynamics on LiCres efficiency. These results will help design strategies where LiCre can be used to conduct genetic studies at high spatio-temporal resolution, or implement it in industrial and biomedical applications.

bioengineering↗

bioRxiv2022

Production of recombinant heterotrimeric mini-procollagen I and homotrimeric II mini-procollagen II reveals new cleavage sites for BMP-1

The proteolytic conversion of soluble procollagens into mature collagen monomers is a critical step to decrease their solubility and trigger collagen fibril formation. In the case of collagens I, II and III, this maturation process is driven by several extracellular metalloproteinases such as BMP-1, tolloid-like proteinases, meprin , meprin {beta}, ADAMTS-2 and ADAMTS-14 but the extensive characterization of these proteolytic events has been hampered by the lack of recombinant procollagens. We previously reported the production and partial characterization of recombinant homotrimeric proteins derived from procollagen III (mini-procollagens III) and, in this study, we describe how we have extended this previous work to the production of heterotrimeric mini-procollagen I and homotrimeric mini-procollagen II. These mini-procollagens include truncated triple helices and intact C-telopeptide and C-propeptide domains and were produced in suspension in HEK293-F cells with yields ranging from 2.5 mg/L to 10 mg/L after purification. They proved very useful tools to analyze the effect of calcium on the stability of the procollagen C-terminal region and to compare the procollagen C-proteinase activity of BMP-1 on the three major fibrillar procollagens or their ability to interact with various partners such as PCPE-1. Using mass spectrometry to map BMP-1 cleavage sites on the mini-procollagens, we confirmed all previously described sites but also revealed two additional cleavage sites in the 1 chain of procollagens I and II. This result shows that the mini-procollagen toolkit offers a broad range of perspectives to make functional studies but also possibly structural analyses or to develop drug screening assays.

biochemistry↗

bioRxiv2021

Procollagen C-Proteinase Enhancer 1 (PCPE-1) is a marker of myocardial fibrosis and impaired cardiac function in a murine model of pressure overload

(1) AimsProcollagen C-proteinase enhancer 1 (PCPE-1) is an extracellular matrix protein and a major regulator of fibrillar collagen biosynthesis. Previous work has shown that its abundance is often increased in the context of tissue repair and fibrosis. The present study was designed to evaluate its potential as a biomarker of myocardial interstitial fibrosis (MIF), a well-established pathogenic pathway leading to heart failure. (2) Methods and ResultsCardiac fibrosis was induced in rats using an optimized model of chronic pressure overload triggered by angiotensin II and N{omega}-nitro-L-arginine methyl ester (L-NAME). All treated animals suffered from heart hypertrophy and the increase in heart collagen volume fraction (CVF), evidenced by histology and 68Ga-Collagelin uptake, confirmed the development of cardiac fibrosis. Functional analysis by simultaneous PET-MRI further showed that our model closely reflected the pathological features seen in human MIF, including left ventricle thickening and diastolic dysfunction associated with decreased ejection fraction. PCPE-1 mRNA and protein levels were augmented by factors of 3.4 and 6.1 respectively in the heart tissue of treated rats. Moreover, protein abundance was well-correlated with CVF (r=0.92, p<0.0001) and PCPE-1 immuno-detection mainly localized the protein to fibrotic areas. Finally, PCPE-1 plasma levels measured by ELISA were increased in fibrotic rats compared to controls. (3) ConclusionTogether, our findings demonstrate that PCPE-1 levels in the heart and circulation tightly reflect the cardiac fibrosis status and heart function impairment in rats and suggest that it could be a very useful marker to monitor human heart diseases leading to fibrosis.

physiology↗

bioRxiv2020

Cathepsin D exacerbates SPARC-driven aggressiveness by limited proteolysis in triple-negative breast cancer

Extracellular matrix (ECM) remodeling by proteases results in the release of protein fragments that promote tumor progression and metastasis. The protease cathepsin D (cath-D), a marker of poor prognosis in triple-negative breast cancer (TNBC), is aberrantly secreted in the tumor microenvironment. Using degradomic analyses by TAILS, we discovered that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited proteolysis of SPARC, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments.

cancer biology↗