Structural insights into regulation of CCN protein activities and functions.

CCN proteins play important functions during development, in repair mechanisms following tissue injury, as well as in pathophysiologic mechanisms of metastasis of cancer. CCNs are secreted proteins that have a multimodular structure and are categorized as matricellular proteins. Although the prevailing view is that CCN proteins regulate biologic processes by interacting with a wide array of other proteins in the microenvironment of the extracellular matrix, the molecular mechanisms of action of CCN proteins are still poorly understood. Not dissuading the current view, however, the recent appreciation that these proteins are signaling proteins in their own right and may even be considered preproproteins controlled by endopeptidases to release a C-terminal bioactive peptide has opened new avenues of research. Also, the recent resolution of the crystal structure of two of the domains of CCN3 have provided new knowledge with implications for the entire CCN family. These resolved structures in combination with structural predictions based upon the AlphaFold artificial intelligence tool provide means to shed new light on CCN functions in context of the notable literature in the field. CCN proteins have emerged as important therapeutic targets in several disease conditions, and clinical trials are currently ongoing. Thus, a review that critically discusses structure - function relationship of CCN proteins, in particular as it relates to interactions with other proteins in the extracellular milieu and on the cell surface, as well as to cell signaling activities of these proteins, is very timely. Suggested mechanism for activation and inhibition of signaling by the CCN protein family (graphics generated with BioRender.com ).

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation.

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.

DHA-induced stress response in human colon cancer cells - Focus on oxidative stress and autophagy.

Polyunsaturated fatty acids (PUFAs) are important constituents of the diet and health benefits of omega-3/n-3 PUFAs, especially eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) have been well documented in relation to several diseases. Increasing evidence suggests that n-3 PUFAs may have anticancer activity and improve the effect of conventional cancer therapy. The mechanisms behind these effects are still unclear and need to be elucidated. We have examined the DHA-induced stress response in two human colon cancer cell lines, SW620 and Caco-2. SW620 cells are growth-inhibited at early time points by DHA, while the growth of Caco-2 cells almost remains unaffected by the same treatment. Gene expression analysis of SW620 cells treated with DHA revealed changes at early time points; transcripts involved in oxidative stress and autophagy were among the first to be differentially expressed. We find that oxidative stress is induced in both cell lines, although at different time points and to different extent. DHA induced nuclear translocation of the oxidative stress sensor NFE2L2 in both cell lines, indicating an induction of an anti-oxidative response. However, vitamin E did not counteract ROS-production or the translocation of NFE2L2 to the nucleus. Neither vitamin E nor the antioxidants butylated hydoxyanisole (BHA) and butylated hydoxytoluene (BHT) did affect the growth inhibition in SW620 cells after DHA-treatment. Also, siRNA-mediated down-regulation of NFE2L2 did not sensitize SW620 and Caco-2 cells to DHA. These results indicate that oxidative stress response is not the cause of DHA-induced cytotoxicity in SW620 cells. Using biochemical and imaging based functional assays, we found a low basal level of autophagy and no increase in autophagic flux after adding DHA to the SW620 cells. However, Caco-2 cells displayed a higher level of autophagy, both in the absence and presence of DHA. Inhibition of autophagy by siRNA mediated knock down of ATG5 and ATG7 sensitized both SW620 and Caco-2 cells to DHA. Stimulation of autophagy by rapamycin in SW620 and Caco-2 cells resulted in decreased DHA-sensitivity and inhibition of autophagy in Caco-2 cells by chloroquine resulted in increased DHA-sensitivity. These results suggest that autophagy is important for the DHA sensitivity of colon cancer cells and imply possible therapeutic effects of this fatty acid against cancer cells with low autophagy.

The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells.

Accumulation and aggregation of misfolded proteins is a hallmark of several diseases collectively known as proteinopathies. Autophagy has a cytoprotective role in diseases associated with protein aggregates. Age-related macular degeneration (AMD) is the most common neurodegenerative eye disease that evokes blindness in elderly. AMD is characterized by degeneration of retinal pigment epithelial (RPE) cells and leads to loss of photoreceptor cells and central vision. The initial phase associates with accumulation of intracellular lipofuscin and extracellular deposits called drusen. Epidemiological studies have suggested an inverse correlation between dietary intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the risk of developing neurodegenerative diseases, including AMD. However, the disease-preventive mechanism(s) mobilized by n-3 PUFAs is not completely understood. In human retinal pigment epithelial cells we find that physiologically relevant doses of the n-3 PUFA docosahexaenoic acid (DHA) induce a transient increase in cellular reactive oxygen species (ROS) levels that activates the oxidative stress response regulator NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2). Simultaneously, there is a transient increase in intracellular protein aggregates containing SQSTM1/p62 (sequestosome 1) and an increase in autophagy. Pretreatment with DHA rescues the cells from cell cycle arrest induced by misfolded proteins or oxidative stress. Cells with a downregulated oxidative stress response, or autophagy, respond with reduced cell growth and survival after DHA supplementation. These results suggest that DHA both induces endogenous antioxidants and mobilizes selective autophagy of misfolded proteins. Both mechanisms could be relevant to reduce the risk of developing aggregate-associate diseases such as AMD.

Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans.

3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/ß domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket.

Divergent ß-hairpins determine double-strand versus single-strand substrate recognition of human AlkB-homologues 2 and 3.

Human AlkB homologues ABH2 and ABH3 repair 1-methyladenine and 3-methylcytosine in DNA/RNA by oxidative demethylation. The enzymes have similar overall folds and active sites, but are functionally divergent. ABH2 efficiently demethylates both single- and double-stranded (ds) DNA, whereas ABH3 has a strong preference for single-stranded DNA and RNA. We find that divergent F1 ß-hairpins in proximity of the active sites of ABH2 and ABH3 are central for substrate specificities. Swapping F1 hairpins between the enzymes resulted in hybrid proteins resembling the donor proteins. Surprisingly, mutation of the intercalating residue F102 had little effect on activity, while the double mutant V101A/F102A was catalytically impaired. These residues form part of an important hydrophobic network only present in ABH2. In this functionally important network, F124 stacks with the flipped out base while L157 apparently functions as a buffer stop to position the lesion in the catalytic pocket for repair. F1 in ABH3 contains charged and polar residues preventing use of dsDNA substrate. Thus, E123 in ABH3 corresponds to F102 in ABH2 and the E123F-variant gained capacity to repair dsDNA with no loss in single strand repair capacity. In conclusion, divergent sequences outside of the active site determine substrate specificities of ABH2 and ABH3.