A molecular landscape of quiescence and proliferation highlights the role of Pten in mammary gland acinogenesis.

Cell context is key for cell state. Using physiologically relevant models of laminin-rich extracellular matrix (lrECM) induction of mammary epithelial cell quiescence and differentiation, we provide a landscape of the key molecules for the proliferation-quiescence decision, identifying multiple layers of regulation at the mRNA and protein levels. Quiescence occurred despite activity of Fak (also known as PTK2), Src and phosphoinositide 3-kinases (PI3Ks), suggesting the existence of a disconnecting node between upstream and downstream proliferative signalling. Pten, a lipid and protein phosphatase, fulfils this role, because its inhibition increased proliferation and restored signalling via the Akt, mTORC1, mTORC2 and mitogen-activated protein kinase (MAPK) pathways. Pten and laminin levels were positively correlated in developing murine mammary epithelia, and Pten localized apicolaterally in luminal cells in ducts and near the nascent lumen in terminal end buds. Consistently, in three-dimensional acinogenesis models, Pten was required for triggering and sustaining quiescence, polarity and architecture. The multilayered regulatory circuitry that we uncovered provides an explanation for the robustness of quiescence within a growth-suppressive microenvironment, which could nonetheless be disrupted by perturbations in master regulators such as Pten.

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM), a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM) displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB) cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling.

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.

Extracellular matrix stiffness regulates degradation of MST2 via SCF ßTrCP.

The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFßTrCP E3 ubiquitin ligase and silencing ßTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that ßTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.

Identificação das proteínas interagentes de Yes-Associated protein (YAP), um efetor da via Hippo, em células epiteliais mamárias expostas à matriz extracelular rica em laminina / Identification of interacting proteins of Yes-Associated Protein (Yap) in mammary epithelial cells exposed to laminin-rich extracellular matrix

A sinalização da matriz extracelular (MEC) é essencial para a determinação do destino e comportamento de células epiteliais da glândula mamária. Entretanto, pouco é conhecido sobre os mecanismos moleculares envolvidos nesse processo. A via Hippo, uma cascata de sinalização que participa da regulação de diversos comportamentos celulares, incluindo o tamanho de órgãos, parece ser uma importante candidata a mediadora sinalização da MEC. Resultados preliminares do laboratório indicam que a arquitetura tecidual e a membrana basal, componente da MEC de epitélios e outros tecidos, influenciam a localização, concentração e atividade de YAP, uma proteína efetora da via Hippo, em células epiteliais mamárias. Neste contexto, o objetivo deste trabalho foi identificar as proteínas que interagem com Yap (ortólogo de YAP em camundongo) nas células epiteliais da glândula mamária em resposta à membrana basal. Foram utilizadas células EpH4, uma linhagem mamária não-tumoral murina, como modelo de diferenciação funcional e formação de ácinos em um ensaio de cultura tridimensional (3D). O tratamento de estruturas multicelulares 3D pré-formadas em placas nãoadesiva com uma matriz rica em laminina (lrECM) alterou a localização e o padrão subcelular de Yap, assim como a expressão gênica de membros da via Hippo e dos alvos de Yap, mas não alterou a expressão das proteínas da via em nível de proteína. O ensaio de co-imunoprecipitação (CoIP) seguida de análise por espectrometria de massas identificou um conjunto diferencial de proteínas que interagem com Yap na fração citoplasmática de células EpH4 cultivadas na ausência ou na presença de lrECM em um modelo de ECM-overlay. Uma análise realizada junto à database KEGG Pathways revelou que os possíveis interagentes Yap nas células cultivadas não-tratadas com lrECM participam de processos relacionados à proteólise mediada por ubiquitina, enquanto nas células expostas à lrECM os possíveis interagentes estão associados a processos metabólicos e são especialmente proteínas-chave do metabolismo de lipídios. A busca na plataforma de redes de interação STRING não identificou trabalhos que destaquem a interação de Yap com estas proteínas. A plataforma Vizit indica a participação de Yap em processos relacionados à síntese e atividade de lipídios e hormônios, o que reforça as evidências de que está pode ser uma nova função de Yap ainda não explorada em detalhes. A fim de se obter resultados complementares à CoIP, padronizamos o ensaio de identificação por biotinilação dependente de proximidade (BioID) em células embrionárias de rim humano da linhagem 293FT. As proteínas isoladas por pulldown foram identificadas por espectrometria de massas e uma análise junto à database Gene Ontology indicou que os possíveis interagentes de Yap nestas células são em sua maioria proteínas relacionadas à via Hippo, o que reforça a robustez do ensaio. Nós pretendemos transpor este sistema para as células EpH4. A expectativa é que, em conjunto, estes resultados nos orientem em projetos futuros para compreender os mecanismos de sinalização da MEC na morfogênese e diferenciação da glândula mamária

Extracellular matrix (ECM)-signaling is crucial for determination of epithelial cell fate and behavior in the mammary gland. However, little is known about the molecular mechanisms involved in these processes. The Hippo pathway, a signaling cascade involved in the regulation of several cellular processes, including organ size, seems to be an important candidate as a mediator of this signaling. Our preliminary results indicate that the tissue architecture and the basement membrane, an ECM component of epithelia and other tissues, influence the location, level and activity of YAP, an effector of the Hippo pathway. In this context, the goal of this work was to identify the proteins that interact with Yap (ortholog of YAP in mouse) in mammary epithelial cells in response to the basement membrane. We used EpH4 cells, a nontumoral murine mammary cell, in a functional differentiation and acini-forming in tridimensional (3D) culture assay. Treatment of 3D multicellular structures pre-formed on nonadhesive plates with a laminin-rich extracellular matrix (lrECM) altered the subcellular localization and pattern of Yap, as well as gene expression of Hippo pathway proteins and Yap targets, but did not altered the expression of the pathway members at the protein level. Coimmunoprecipitation (CoIP) followed by mass spectrometry analysis identified a differential set of proteins interacting with Yap in cytoplasmic fractions of EpH4 cells in the absence or presence of lrECM in an ECM-overlay culture model. An analysis performed with the KEGG Pathways database revealed that putative Yap interactors in non-treated cells participate in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to lrECM Yap interactors are associated to metabolic processes and are mainly key-proteins of metabolism of lipids and carbohydrates. A search in interaction networks platform STRING did not identify previous works that showing the interaction of Yap with these proteins. Vizit platform indicated the participation of Yap in processes related to the synthesis and activity of lipids and hormones, which reinforces the evidences that Yap can play a novel poorly explored role. To obtain complementary results to CoIP, we devised the proximity-dependent biotinylation identification (BioID) assay on embryonic renal cells of 293FT cell line. Pulldown-isolated proteins were identified by mass spectrometry and an analysis performed with Gene Ontology database revealed that putative Yap interactors are Hippo pathway-related proteins, which reinforces the robustness of the assay. We intend to transpose this system to the EpH4 cells. We expect that, together, these results will guide us in future projects to understand the signaling mechanisms of ECM in mammary gland morphogenesis and differentiation