Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system.

The bacterial human pathogen Helicobacter pylori produces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. The cagT4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shape N-terminal domains (CagIN) prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagIC with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagIC were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagIC plays a key role in cagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cagT4SS, a crucial risk factor for gastric cancer development.

Identification of a two-component regulatory system involved in antimicrobial peptide resistance in Streptococcus pneumoniae.

Two-component regulatory systems (TCS) are among the most widespread mechanisms that bacteria use to sense and respond to environmental changes. In the human pathogen Streptococcus pneumoniae, a total of 13 TCS have been identified and many of them have been linked to pathogenicity. Notably, TCS01 strongly contributes to pneumococcal virulence in several infection models. However, it remains one of the least studied TCS in pneumococci and its functional role is still unclear. In this study, we demonstrate that TCS01 cooperates with a BceAB-type ABC transporter to sense and induce resistance to structurally-unrelated antimicrobial peptides of bacterial origin that all target undecaprenyl-pyrophosphate or lipid II, which are essential precursors of cell wall biosynthesis. Even though tcs01 and bceAB genes do not locate in the same gene cluster, disruption of either of them equally sensitized the bacterium to the same set of antimicrobial peptides. We show that the key function of TCS01 is to upregulate the expression of the transporter, while the latter appears the main actor in resistance. Electrophoretic mobility shift assays further demonstrated that the response regulator of TCS01 binds to the promoter region of the bceAB genes, implying a direct control of these genes. The BceAB transporter was overexpressed and purified from E. coli. After reconstitution in liposomes, it displayed substantial ATPase and GTPase activities that were stimulated by antimicrobial peptides to which it confers resistance to, revealing new functional features of a BceAB-type transporter. Altogether, this inducible defense mechanism likely contributes to the survival of the opportunistic microorganism in the human host, in which competition among commensal microorganisms is a key determinant for effective host colonization and invasive path.

The basement membrane in epidermal polarity, stemness, and regeneration.

The epidermis is a specialized epithelium that constitutes the outermost layer of the skin, and it provides a protective barrier against environmental assaults. Primarily consisting of multilayered keratinocytes, the epidermis is continuously renewed by proliferation of stem cells and the differentiation of their progeny, which undergo terminal differentiation as they leave the basal layer and move upward toward the surface, where they die and slough off. Basal keratinocytes rest on a basement membrane at the dermal-epidermal junction that is composed of specific extracellular matrix proteins organized into interactive and mechanically supportive networks. Firm attachment of basal keratinocytes, and their dynamic regulation via focal adhesions and hemidesmosomes, is essential for maintaining major skin processes, such as self-renewal, barrier function, and resistance to physical and chemical stresses. The adhesive integrin receptors expressed by epidermal cells serve structural, signaling, and mechanosensory roles that are critical for epidermal cell anchorage and tissue homeostasis. More specifically, the basement membrane components play key roles in preserving the stem cell pool, and establishing cell polarity cues enabling asymmetric cell divisions, which result in the transition from a proliferative basal cell layer to suprabasal cells committed to terminal differentiation. Finally, through a well-regulated sequence of synthesis and remodeling, the components of the dermal-epidermal junction play an essential role in regeneration of the epidermis during skin healing. Here too, they provide biological and mechanical signals that are essential to the restoration of barrier function.

Altered Distribution and Expression of Syndecan-1 and -4 as an Additional Hallmark in Psoriasis.

Syndecans act as independent co-receptors to exert biological activities and their altered function is associated with many pathophysiological conditions. Here, syndecan-1 and -4 were examined in lesional skin of patients with psoriasis. Immunohistochemical staining confirmed altered syndecan-1 distribution and revealed absence of syndecan-4 expression in the epidermis. Fibronectin (FN)-known to influence inflammation and keratinocyte hyperproliferation via α5ß1 integrin in psoriasis-was also decreased. Syndecan-1 and -4 expression was analyzed in freshly isolated lesional psoriatic human keratinocytes (PHK) characterized based on their proliferation and differentiation properties. mRNA levels of syndecan-1 were similar between healthy and PHK, while syndecan-4 was significantly decreased. Cell growth and release of the pro-inflammatory Tumor Necrosis Factor-alpha (TNFα) were selectively and significantly induced in PHKs plated on FN. Results from co-culture of healthy keratinocytes and psoriatic fibroblasts led to the speculation that at least one factor released by fibroblasts down-regulate syndecan-1 expression in PHK plated on FN. To assay if biological treatments for psoriasis target keratinocyte proliferation, gelatin-based patches enriched with inteleukin (IL)-17α or TNFα blockers were prepared and tested using a full-thickness healthy epidermal model (Phenion®). Immunohistochemistry analysis showed that both blockers impacted the localisation of syndecan-1 within the refined epidermis. These results provide evidence that syndecans expression are modified in psoriasis, suggesting that they may represent markers of interest in this pathology.

Laminin 332 in cancer: When the extracellular matrix turns signals from cell anchorage to cell movement.

Laminin 332 is crucial in the biology of epithelia. This large extracellular matrix protein consists of the heterotrimeric assembly of three subunits - α3, ß3, and γ2 - and its multifunctionality relies on a number of extracellular proteolytic processing events. Laminin 332 is central to normal epithelium homeostasis by sustaining cell adhesion, polarity, proliferation, and differentiation. It also supports a major function in epithelial tissue formation, repair, and regeneration by buttressing cell migration and survival and basement membrane assembly. Interest in this protein increased after the discovery that its expression is perturbed in tumor cells, cancer-associated fibroblasts, and the tumor microenvironment. This review summarizes current knowledge regarding the established involvement of the laminin 332 γ2 chain in tumor invasiveness and discusses the role of its α3 and ß3 subunits.

In vitro epidermis model mimicking IGF-1-specific age-related decline.

Ageing is a complex multifaceted process affecting skin functionality and structure. Several 3D organotypic skin culture models have reproduced ageing by inducing replicative senescence, glycation or oxidative stress. Yet, very few models have focused on hormonal ageing and especially the insulin-like growth factor 1 (IGF-1) signalling pathway, which has been associated with longevity in animal studies and is necessary for the early stages of skin development. In this study, we built an organotypic epidermis model with targeted IGF-1 receptor knockdown to reproduce some aspects of hormonal ageing on skin. Our model displayed morphological and functional features of aged epidermis, which were mostly attributed to a loss of function of the Stratum basale. IGF-1 receptor knockdown keratinocytes depicted an extended cell cycle, reduced proliferation potential and reduced adhesion capacities and greater sensitivity to oxidative stress than control cells. Altogether, this model represents an essential tool for further investigations into the mechanisms linked to some aspects of hormonal decline or when screening for potent anti-ageing compounds.

Dermal stiffness governs the topography of the epidermis and the underlying basement membrane in young and old human skin.

The epidermis is a stratified epithelium that forms the outer layer of the skin. It is composed primarily of keratinocytes and is constantly renewed by the proliferation of stem cells and their progeny that undergo terminal differentiation as they leave the basal layer and migrate to the skin surface. Basal keratinocytes rest on a basement membrane composed of an extracellular matrix that controls their fate via integrin-mediated focal adhesions and hemidesmosomes which are critical elements of the epidermal barrier and promote its regenerative capabilities. The distribution of basal cells with optimal activity provides the basement membrane with its characteristic undulating shape; this configuration disappears with age, leading to epidermal weakness. In this study, we present an in-depth imaging analysis of basal keratinocyte anchorage in samples of human skin from participants across the age spectrum. Our findings reveal that skin aging is associated with the depletion of hemidesmosomes that provide crucial support for stem cell maintenance; their depletion correlates with the loss of the characteristic basement membrane structure. Atomic force microscopy studies of skin and in vitro experiments revealed that the increase in tissue stiffness observed with aging triggers mechanical signals that alter the basement membrane structure and reduce the extent of basal keratinocyte anchorage, forcing them to differentiate. Genomic analysis revealed that epidermal aging was associated with mechanical induction of the transcription factor Krüppel-like factor 4. The altered mechanical properties of tissue being a new hallmark of aging, our work opens new avenues for the development of skin rejuvenation strategies.

Cell surface proteoglycans syndecan-1 and -4 bind overlapping but distinct sites in laminin α3 LG45 protein domain.

Keratinocyte migration during epidermal repair depends on interactions between cellular heparan sulfate proteoglycan receptors, syndecan-1 and -4, and the C-terminal globular domains (LG45) of the extracellular matrix protein laminin 332. This study investigates the molecular basis of the binding specificity of the syndecan-1 and -4 receptors expressed by human keratinocytes. We used site-directed mutagenesis to alter a recombinant LG45 protein by substituting the most critical basic residues with glutamine. All proteins were expressed in mammalian cells, purified, and characterized biochemically. We used in vitro binding assays, including surface plasmon resonance, to examine interactions between mutated LG45 and heparan sulfates, syndecan-1 and -4. We identify a major heparin binding domain on the outer edge of a ß-strand of LG45 surrounded by a track of converging low affinity residues. This domain harbors distinctive syndecan-1 and -4 binding-specific sequences. This is the first study to demonstrate a binding specificity of two proteoglycans produced by a single cell type. In addition, we found that although syndecan-1 interacts exclusively through its glycosaminoglycan chains, syndecan-4 binding relies on both its core protein and its heparan sulfate chains. These results suggest that LG45 may trigger different signals toward keratinocytes depending on its interaction with syndecan-1 or -4.

Palladium-catalyzed direct arylation of polysubstituted benzofurans.

An efficient access to 2-substituted 3-arylbenzofurans through a palladium-catalyzed C3 direct arylation of 2-substituted benzofurans with aryl bromides is described. The scope and limitation of this reaction was studied. The method tolerates a variety of functional groups on the aryl halide and has been successfully extended to polysubstituted benzofurans to obtain the corresponding 3-arylbenzofurans with good to excellent yields.

Melanoma cells produce multiple laminin isoforms and strongly migrate on α5 laminin(s) via several integrin receptors.

Melanoma cells express and interact with laminins (LMs) and other basement membrane components during invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of laminin isoforms in melanoma. Immunohistochemistry of melanoma specimens and immunoprecipitation/western blotting of melanoma cell lines indicated expression of laminin-111/121, laminin-211, laminin-411/421, and laminin-511/521. Laminin-332 was not detected. In functional assays, laminin-111, laminin-332, and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic cell migration either constitutively or following stimulation with insulin-like growth factors. Both placenta and recombinant laminin-511 preparations were highly active, and the isolated recombinant IVa domain of LMα5 also promoted cell migration. Function-blocking antibodies in cell migration assays revealed α6ß1 integrin as the major receptor for laminin-111, and both α3ß1 and α6ß1 integrins for laminin-332 and laminin-511. In contrast, isolated LMα5 IVa domain-promoted melanoma cell migration was largely mediated via αVß3 integrin and inhibited by RGD peptides. Given the ubiquitous expression of α5 laminins in melanoma cells and in melanoma-target tissues/anatomical structures, as well as the strong migration-promoting activity of these laminin isoforms, the α5 laminins emerge as putative primary extracellular matrix mediators of melanoma invasion and metastasis via α3ß1 and other integrin receptors.

Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry.

A striking feature of skin organization is that the extracellular matrix (ECM) occupies a larger volume than the cells. Skin ECM also directly contributes to aging and most cutaneous diseases. In recent years, specific ECM enrichment protocols combined with in silico approaches allowed the proteomic description of the matrisome of various organs and tumor samples. Nevertheless, the skin matrisome remains under-studied and protocols allowing the efficient recovery of the diverse ECM found in skin are still to be described. Here, we compared four protocols allowing the enrichment of ECM proteins from adult mouse back skin and found that all protocols led to a significant enrichment (up to 65%) of matrisome proteins when compared to total skin lysates. The protocols based on decellularization and solubility profiling gave the best results in terms of numbers of proteins identified and confirmed that skin matrisome proteins exhibit very diverse solubility and abundance profiles. We also report the first description of the skin matrisome of healthy adult mice that includes 236 proteins comprising 95 core matrisome proteins and 141 associated matrisome proteins. These results provide a reliable basis for future characterizations of skin ECM proteins and their dysregulations in disease-specific contexts. SIGNIFICANCE: Extracellular matrix proteins are key players in skin physiopathology and have been involved in several diseases such as genetic disorders, wound healing defects, scleroderma and skin carcinoma. However, skin ECM proteins are numerous, diverse and challenging to analyze by mass spectrometry due to the multiplicity of their post-translational modifications and to the heterogeneity of their solubility profiles. Here, we performed the thorough evaluation of four ECM enrichment protocols compatible with the proteomic analysis of mouse back skin and provide the first description of the adult mouse skin matrisome in homeostasis conditions. Our work will greatly facilitate the future characterization of skin ECM alterations in preclinical mouse models and will inspire new optimizations to analyze the skin matrisome of other species and of human clinical samples.

Benefit of coupling heparin to crosslinked collagen I/III scaffolds for human dermal fibroblast subpopulations' tissue growth.

Currently, there is a lack of models representing the skin dermal heterogeneity for relevant research and skin engineering applications. This is the first study reporting production of dermal equivalents reproducing features of papillary and reticular dermal compartments. Inspired from our current knowledge on the architecture and composition differences between the papillary and reticular dermis, we evaluated different collagen-based porous materials to serve as scaffolds for the three-dimensional expansion of freshly isolated papillary and/or reticular fibroblasts. The scaffolds, composed of either collagen I or collagen I and III mixtures, were prepared by lyophilization. Pore size and hydrolytic stability were controlled by crosslinking with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or EDC/NHS with covalently bound heparin. The evaluation of the resultant "papillary" and "reticular" dermal equivalents was based on the analysis of characteristic features of each dermal compartment, such as cell density and deposition of newly synthetized extracellular matrix components in histological sections. Crosslinking supported cell growth during dermal tissue formation independent on the fibroblast subpopulation. The presence of collagen III seemed to have some positive but non-specific effect only on the maintenance of the mechanical strength of the scaffolds during dermal formation. Histological analyses demonstrated a significant and specific effect of heparin on generating dermal equivalents reproducing the respective higher papillary than reticular cell densities and supporting distinct extracellular matrix components deposition (three to five times more carbohydrate material deposited by papillary fibroblasts in all scaffolds containing heparin, while higher collagen production was observed only in the presence of heparin).

Incidence of an Intracellular Multiplication Niche among Acinetobacter baumannii Clinical Isolates.

The spread of antibiotic-resistant Acinetobacter baumannii poses a significant threat to public health worldwide. This nosocomial bacterial pathogen can be associated with life-threatening infections, particularly in intensive care units. A. baumannii is mainly described as an extracellular pathogen with restricted survival within cells. This study shows that a subset of A. baumannii clinical isolates extensively multiply within nonphagocytic immortalized and primary cells without the induction of apoptosis and with bacterial clusters visible up to 48 h after infection. This phenotype was observed for the A. baumannii C4 strain associated with high mortality in a hospital outbreak and the A. baumannii ABC141 strain, which was isolated from the skin but was found to be hyperinvasive. Intracellular multiplication of these A. baumannii strains occurred within spacious single membrane-bound vacuoles, labeled with the lysosomal associate membrane protein (LAMP1). However, these compartments excluded lysotracker, an indicator of acidic pH, suggesting that A. baumannii can divert its trafficking away from the lysosomal degradative pathway. These compartments were also devoid of autophagy features. A high-content microscopy screen of 43 additional A. baumannii clinical isolates highlighted various phenotypes, and (i) the majority of isolates remained extracellular, (ii) a significant proportion was capable of invasion and limited persistence, and (iii) three more isolates efficiently multiplied within LAMP1-positive vacuoles, one of which was also hyperinvasive. These data identify an intracellular niche for specific A. baumannii clinical isolates that enables extensive multiplication in an environment protected from host immune responses and out of reach of many antibiotics. IMPORTANCE Multidrug-resistant Acinetobacter baumannii isolates are associated with significant morbidity and mortality in hospitals worldwide. Understanding their pathogenicity is critical for improving therapeutic management. Although A. baumannii can steadily adhere to surfaces and host cells, most bacteria remain extracellular. Recent studies have shown that a small proportion of bacteria can invade cells but present limited survival. We have found that some A. baumannii clinical isolates can establish a specialized intracellular niche that sustains extensive intracellular multiplication for a prolonged time without induction of cell death. We propose that this intracellular compartment allows A. baumannii to escape the cell's normal degradative pathway, protecting bacteria from host immune responses and potentially hindering antibiotic accessibility. This may contribute to A. baumannii persistence, relapsing infections, and enhanced mortality in susceptible patients. A high-content microscopy-based screen confirmed that this pathogenicity trait is present in other clinical A. baumannii isolates. There is an urgent need for new antibiotics or alternative antimicrobial approaches, particularly to combat carbapenem-resistant A. baumannii. The discovery of an intracellular niche for this pathogen, as well as hyperinvasive isolates, may help guide the development of antimicrobial therapies and diagnostics in the future.

Synthesis of 3-aryl-2-arylamidobenzofurans based on the Curtius rearrangement.

The synthesis of novel 3-aryl-2-arylamidobenzofurans has been accomplished via a Curtius rearrangement strategy in four steps from benzofuran-2-carboxylic acids. The requisite Suzuki-Miyaura cross-coupling, with benzyl 3-bromobenzofuran-2-ylcarbamate or 2-arylamido-3-bromobenzofurans, revealed an unusual reductive debromination process due to the presence of the free NH group. This dehalogenation can be suppressed by N-alkylation. DMAP is an efficient reagent for the one-pot conversion of benzyl benzofuran-2-ylcarbamates into the corresponding benzofuran-2-arylamides through aroylation, thus acting both as an acyl transfer reagent and a deprotecting agent of the Cbz group. A mechanism is postulated.

Human mast cells adhere to and migrate on epithelial and vascular basement membrane laminins LM-332 and LM-511 via alpha3beta1 integrin.

Mast cells (MCs) are multifunctional effectors of the immune system that are distributed in many tissues, often in close association with the basement membrane of blood vessels, epithelium and nerves. Laminins (LMs), a family of large alphabetagamma heterotrimeric proteins, are major components of basement membrane that strongly promote cell adhesion and migration. In this study, we investigated the role of LM isoforms and their integrin receptors in human MC biology in vitro. In functional assays, alpha3-(LM-332) and alpha5-(LM-511) LMs, but not alpha1-(LM-111), alpha2-(LM-211), or alpha4-(LM-411) LMs, readily promoted adhesion and migration of cultured MCs. These activities were strongly enhanced by various stimuli. alpha3-LM was also able to costimulate IL-8 production. Among LM-binding integrins, MCs expressed alpha(3)beta(1), but not alpha(6)beta(1), alpha(7)beta(1), or alpha(6)beta(4), integrins. Blocking Abs to alpha(3)beta(1) integrin caused inhibition of both cell adhesion and migration on alpha3- and alpha5-LMs. Immunohistochemical studies on skin showed that MCs colocalized with epithelial and vascular basement membranes that expressed alpha3- and alpha5-LMs and that MCs expressed alpha(3) integrin but not alpha(6) integrin(s). These results demonstrate a role for alpha3- and alpha5-LMs and their alpha(3)beta(1) integrin receptor in MC biology. This may explain the intimate structural and functional interactions that MCs have with specific basement membranes.

Extracellular matrix-based cancer targeting.

Tumor extracellular matrix (ECM) operates in a coordinated mode with cancer and stroma cells to evoke the multistep process of metastatic potential. The remodeled tumor-associated matrix provides a point for direct or complementary therapeutic targeting. Here, we cover and critically address the importance of ECM networks and their macromolecules in cancer. We focus on the roles of key structural and functional ECM components, and their degradation enzymes and extracellular vesicles, aiming at improving our understanding of the mechanisms contributing to tumor initiation, growth, and dissemination, and discuss potential new approaches for ECM-based therapeutic targeting and diagnosis.

Validation of an implantable bioink using mechanical extraction of human skin cells: First steps to a 3D bioprinting treatment of deep second degree burn.

Clinical grade cultured epithelial autograft (CEA) are routinely used to treat burns covering more than 60% of the total body surface area. However, although the epidermis may be efficiently repaired by CEA, the dermal layer, which is not spared in deep burns, requires additional treatment strategies. Our aim is to develop an innovative method of skin regeneration based on in situ 3D bioprinting of freshly isolated autologous skin cells. We describe herein bioink formulation and cell preparation steps together with experimental data validating a straightforward enzyme-free protocol of skin cell extraction. This procedure complies with both the specific needs of 3D bioprinting process and the stringent rules of good manufacturing practices. This mechanical extraction protocol, starting from human skin biopsies, allows harvesting a sufficient amount of both viable and growing keratinocytes and fibroblasts. We demonstrated that a dermis may be reconstituted in vitro starting from a medical grade bioink and mechanically extracted skin cells. In these experiments, proliferation of the extracted cells can be observed over the first 21 days period after 3D bioprinting and the analysis of type I collagen exhibited a de novo production of extracellular matrix proteins. Finally, in vivo experiments in a murine model of severe burn provided evidences that a topical application of our medical grade bioink was feasible and well-tolerated. Overall, these results represent a valuable groundwork for the design of future 3D bioprinting tissue engineering strategies aimed at treating, in a single intraoperative step, patients suffering from extended severe burns.

Regulated laminin-332 expression in human islets of Langerhans.

Laminin-332 (LN-332) is a basement membrane component known to exert a beneficial effect on rat pancreatic beta cells in vitro. In this work, we analyzed the expression of LN-332 in human islets, its expression after inflammatory insults by cytokines, and the molecular mechanisms responsible for this effect. By Western blotting and RT-PCR, we showed that LN-332 was expressed in isolated human islets. By immunofluorescence on pancreas sections, we observed that labeling was confined to endocrine cells in islets. Confocal microscopy analysis on isolated islet cells revealed that labeling was granular but did not colocalize with hormone secretory granules. LN-332 was most abundant in cultured islets compared to freshly isolated islets and was found in culture medium, which suggests that it was secreted by islets. When islets were exposed to interleukin (IL)-1beta, expression and secretion of LN-332 increased as compared to control. No effect was observed with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited culture- and IL-1beta-induced LN-332 expression in islets. These results show that LN-332, known to have some beneficial effect on beta cells in vitro, is produced and secreted by endocrine islet cells and is up-regulated by stressing conditions such as culture and IL-1beta-exposure.

The Human Epidermal Basement Membrane: A Shaped and Cell Instructive Platform That Aging Slowly Alters.

One of the most important functions of skin is to act as a protective barrier. To fulfill this role, the structural integrity of the skin depends on the dermal-epidermal junction-a complex network of extracellular matrix macromolecules that connect the outer epidermal layer to the underlying dermis. This junction provides both a structural support to keratinocytes and a specific niche that mediates signals influencing their behavior. It displays a distinctive microarchitecture characterized by an undulating pattern, strengthening dermal-epidermal connectivity and crosstalk. The optimal stiffness arising from the overall molecular organization, together with characteristic anchoring complexes, keeps the dermis and epidermis layers extremely well connected and capable of proper epidermal renewal and regeneration. Due to intrinsic and extrinsic factors, a large number of structural and biological changes accompany skin aging. These changes progressively weaken the dermal-epidermal junction substructure and affect its functions, contributing to the gradual decline in overall skin physiology. Most changes involve reduced turnover or altered enzymatic or non-enzymatic post-translational modifications, compromising the mechanical properties of matrix components and cells. This review combines recent and older data on organization of the dermal-epidermal junction, its mechanical properties and role in mechanotransduction, its involvement in regeneration, and its fate during the aging process.