StAR‐related lipid transfer domain‐3 (STARD3) is a sterol‐binding protein that creates endoplasmic reticulum (ER)–endosome contact sites. How this protein, at the crossroad between sterol uptake and ...synthesis pathways, impacts the intracellular distribution of this lipid was ill‐defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3‐mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER–endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER‐anchored partner, Vesicle‐associated membrane protein‐associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts. Thus, STARD3 is a cholesterol transporter scaffolding ER–endosome contacts and modulating cellular cholesterol repartition by delivering cholesterol to endosomes.
Synopsis
The cholesterol transfer protein STARD3, which scaffolds ER–endosome contact sites, controls cellular cholesterol distribution by efficiently delivering ER cholesterol to endosomes.
STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane.
STARD3‐mediated cholesterol routing depends on both its START domain‐dependent lipid transfer activity and its ability to create ER–endosome contacts with the ER‐anchored VAP proteins.
In vitro reconstitution assays indicate that STARD3 and VAP assemble into a highly efficient cholesterol transport machine.
The cholesterol transfer protein STARD3, which scaffolds ER–endosome contact sites, controls cellular cholesterol distribution by efficiently delivering ER cholesterol to endosomes.
The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain is a protein module of approximately210 residues that binds lipids, including sterols. Fifteen mammalian ...proteins, STARD1-STARD15, possess a START domain and these can be grouped into six subfamilies. Cholesterol, 25-hydroxycholesterol, phosphatidylcholine, phosphatidylethanolamine and ceramides are ligands for STARD1/STARD3/STARD5, STARD5, STARD2/STARD10, STARD10 and STARD11, respectively. The lipids or sterols bound by the remaining 9 START proteins are unknown. Recent studies show that the C-terminal end of the domain plays a fundamental role, forming a lid over a deep lipid-binding pocket that shields the ligand from the external environment. The START domain can be regarded as a lipid-exchange and/or a lipid-sensing domain. Mammalian START proteins have diverse expression patterns and can be found free in the cytoplasm, attached to membranes or in the nucleus. They appear to function in a variety of distinct physiological processes, such as lipid transfer between intracellular compartments, lipid metabolism and modulation of signaling events. Mutation or misexpression of START proteins is linked to pathological processes, including genetic disorders, autoimmune disease and cancer.
Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP‐A, VAP‐B, and MOSPD2, which interact with proteins at the surface of ...other organelles to build contacts. VAP‐A, VAP‐B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non‐conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non‐conventional FFAT motifs (named Phospho‐FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho‐FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho‐FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER‐endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter‐organelle contacts.
Synopsis
Membrane contact sites (MCS) are subcellular domains formed by close apposition between the membranes of two organelles, which regulate lipid transport and organelle dynamics. Here, phosphorylation of a non‐conventional FFAT motif on Endoplasmic Reticulum (ER) receptor partner proteins is shown to mediate MCS formation and lipid transport.
The ER receptors VAP‐A, VAP‐B and MOSPD2 bind a non‐conventional FFAT motif via their MSP domain.
The alternative FFAT motif (Phospho‐FFAT) is characterized by a key phosphorylated serine/threonine residue.
Structural analyses show that conventional FFATs and alternative phospho‐FFATs bind to VAP differently.
An algorithm to predict Phospho‐FFAT motifs reveals that Phospho‐FFATs are as common as conventional FFATs in the human proteome.
Phosphorylation acts as a switch that activates the ER–endosome MCS formation and sterol exchange.
Phosphorylation of a non‐conventional FFAT motif promotes ER‐endosome membrane contact sites and sterol exchange.
Early hormone-positive breast cancers typically have favorable outcomes, yet long-term surveillance is crucial due to the risk of late recurrences. While many studies associate MMP-11 expression with ...poor prognosis in breast cancer, few focus on early-stage cases. This study explores MMP-11 as an early prognostic marker in hormone-positive breast cancers.
In this retrospective study, 228 women with early hormone-positive invasive ductal carcinoma, treated surgically between 2011 and 2016, were included. MMP-11 expression was measured by immunohistochemistry, and its association with clinical and MRI data was analyzed.
Among the patients (aged 31-89, median 60, with average tumor size of 15.7 mm), MMP-11 staining was observed in half of the cases. This positivity correlated with higher uPA levels and tumor grade but not with nodal status or size. Furthermore, MMP-11 positivity showed specific associations with MRI features. Over a follow-up period of 6.5 years, only 12 oncological events occurred. Disease-free survival was linked to Ki67 and MMP-11.
MMP-11, primarily present in tumor-surrounding stromal cells, correlates with tumor grade and uPA levels. MMP-11 immunohistochemical score demonstrates a suggestive trend in association with disease-free survival, independent of Ki67 and other traditional prognostic factors. This highlights the potential of MMP-11 as a valuable marker in managing early hormone-positive breast cancer.
Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and ...metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the α6β4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM.
We developed new mouse models inducing IEC-specific ablation of α6 integrin either during development (α6
) or in adults (α6
).
Strikingly, all α6
mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of α6 integrin induced in adult mice (α6
) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1β secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation.
We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.
Membrane contact sites (MCSs) are subcellular regions where the membranes of distinct organelles come into close apposition. These specialized areas of the cell, which are involved in inter-organelle ...metabolite exchange, are scaffolded by specific complexes. STARD3 StAR (steroidogenic acute regulatory protein)-related lipid transfer domain-3 and its close paralogue STARD3NL (STARD3 N-terminal like) are involved in the formation of contacts between late-endosomes and the endoplasmic reticulum (ER). The lipid transfer protein (LTP) STARD3 and STARD3NL, which are both anchored on the limiting membrane of late endosomes (LEs), interact with ER-anchored VAP VAMP (vesicle-associated membrane protein)-associated protein (VAP-A and VAP-B) proteins. This direct interaction allows ER-endosome contact formation. STARD3 or STARD3NL-mediated ER-endosome contacts, which affect endosome dynamics, are believed to be involved in cholesterol transport.
Pathological complete response (pCR) after neoadjuvant systemic treatment (NST) is an important prognostic factor in HER2-positive breast cancer. The majority of HER2-positive breast cancers are ...amplified at the HER2 gene locus, several genes are co-amplified with HER2, and a subset of them are co-expressed. The STARD3 gene belongs to the HER2 amplicon, and its role as a predictive marker was never addressed. The objective of this study was to investigate the predictive value of STARD3 protein expression on NST pathological response in HER2-positive breast cancer. In addition, we studied the prognostic value of this marker.
We conducted a retrospective study between 2007 and 2020 on 112 patients with non-metastatic HER2-positive breast cancer treated by NST and then by surgery. We developed an immunohistochemistry assay for STARD3 expression and subcellular localization and determined a score for STARD3-positivity. As STARD3 is an endosomal protein, its expression was considered positive if the intracellular signal pattern was granular.
In this series, pCR was achieved in half of the patients. STARD3 was positive in 86.6% of cases and was significantly associated with pCR in univariate analysis (
= 0.013) and after adjustment on other known pathological parameters (
= 0.044). Performances on pCR prediction showed high sensitivity (96%) and negative predictive value (87%), while specificity was 23% and positive predictive value was 56%. Overall, specific, relapse-free, and distant metastasis-free survivals were similar among STARD3 positive and negative groups, independently of other prognosis factors.
NST is an opportunity for HER2-positive cancers. In this series of over a hundred HER2-positive and non-metastatic patients, a STARD3-negative score was associated with the absence of pathological complete response. This study suggests that determining STARD3 overexpression status on initial biopsies of HER2-positive tumors is an added value for the management of a subset of patients with high probability of no pathological response.
Matrix metalloproteinase 11 (MMP11) is an extracellular proteolytic enzyme belonging to the matrix metalloproteinase (MMP11) family. These proteases are involved in extracellular matrix (ECM) ...remodeling and activation of latent factors. MMP11 is a negative regulator of adipose tissue development and controls energy metabolism in vivo. In cancer, MMP11 expression is associated with poorer survival, and preclinical studies in mice showed that MMP11 accelerates tumor growth. How the metabolic role of MMP11 contributes to cancer development is poorly understood. To address this issue, we developed a series of preclinical mouse mammary gland tumor models by genetic engineering. Tumor growth was studied in mice either deficient (Loss of Function-LOF) or overexpressing MMP11 (Gain of Function-GOF) crossed with a transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV) (MMTV-PyMT). Both GOF and LOF models support roles for MMP11, favoring early tumor growth by increasing proliferation and reducing apoptosis. Of interest, MMP11 promotes Insulin-like Growth Factor-1 (IGF1)/protein kinase B (AKT)/Forkhead box protein O1 (FoxO1) signaling and is associated with a metabolic switch in the tumor, activation of the endoplasmic reticulum stress response, and an alteration in the mitochondrial unfolded protein response with decreased proteasome activity. In addition, high resonance magic angle spinning (HRMAS) metabolomics analysis of tumors from both models established a metabolic signature that favors tumorigenesis when MMP11 is overexpressed. These data support the idea that MMP11 contributes to an adaptive metabolic response, named metabolic flexibility, promoting cancer growth.
The family of StAR related lipid transfer proteins (START) is so-named based on the distinctive capacity for these proteins to transport lipids between membranes. The START domain is a module of ...about 210 residues, which binds lipids such as glycerolipids, sphingolipids and sterols. This domain has a deep lipid-binding pocket – which shields the hydrophic ligand from the external aqueous environment – covered by a lid. Based on their homology, the fifteen START proteins in mammals have been allocated to six distinct subfamilies, each subfamily being more specialized in the transport and/or sensing of a lipid ligand species. However within the same subgroup, their expression profile and their subcellular localization distinguish them and are critical for their different biological functions. Indeed, START proteins act in a variety of distinct physiological processes, such as lipid transfer between intracellular compartments, lipid metabolism and modulation of signaling events. Mutation or deregulated expression of START proteins is linked to pathological processes, including genetic disorders, autoimmune diseases and cancers. Besides the common single START domain, which is always located at the carboxy-terminal end in mammals, most START proteins harbor additional domains predicted to be critical in favoring lipid exchange. Evidence from well characterized START proteins indicates that these additional domains might be tethering machineries able to bring distinct organelles together and create membrane contact sites prone to lipid exchange via the START domain.
•START lipid binding domains are found in 15 human proteins (STARD1-15).•START proteins are involved in intracellular transport of lipids.•Some START proteins tether organelles to create membrane contact sites.•START proteins are implicated in lipid metabolism as well as diseases such as cancer.
Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at ...tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration.
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