A Therapeutic Perspective

AF was funded by a Ph.D. fellowship (PD/BD/135506/2018) from the Fundação para a Ciência e a Tecnologia (FCT) and DB by the FCT Investigator Program (IF/00501/2014/CP1252/CT0001).The Adenosine diphosphate-Ribosylation Factor (ARF) family belongs to the RAS superfamily of small GTPases and is involved in a wide variety of physiological processes, such as cell proliferation, motility and differentiation by regulating membrane traffic and associating with the cytoskeleton. Like other members of the RAS superfamily, ARF family proteins are activated by Guanine nucleotide Exchange Factors (GEFs) and inactivated by GTPase-Activating Proteins (GAPs). When active, they bind effectors, which mediate downstream functions. Several studies have reported that cancer cells are able to subvert membrane traffic regulators to enhance migration and invasion. Indeed, members of the ARF family, including ARF-Like (ARL) proteins have been implicated in tumorigenesis and progression of several types of cancer. Here, we review the role of ARF family members, their GEFs/GAPs and effectors in tumorigenesis and cancer progression, highlighting the ones that can have a pro-oncogenic behavior or function as tumor suppressors. Moreover, we propose possible mechanisms and approaches to target these proteins, toward the development of novel therapeutic strategies to impair tumor progression.publishersversionpublishe

The pursuit of skin pigmentation control mechanisms

Funding: We would like to thank our group for critical reading of the manuscript. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) through projects EXPL/BEXBCM/0379/2013 and PTDC/BIA-CEL/29765/2017. H.M. was supported by a PhD fellowship from FCT (PD/BD/114118/2015), and D.C.B. by the FCT Investigator Program (IF/00501/2014/CP1252/ CT0001). This article is supported by the LYSOCIL project. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 811087.The mechanisms by which the pigment melanin is transferred from melanocytes and processed within keratinocytes to achieve skin pigmentation remain ill-characterized. Nevertheless, several models have emerged in the past decades to explain the transfer process. Here, we review the proposed models for melanin transfer in the skin epidermis, the available evidence supporting each one, and the recent observations in favor of the exo/phagocytosis and shed vesicles models. In order to reconcile the transfer models, we propose that different mechanisms could co-exist to sustain skin pigmentation under different conditions. We also discuss the limited knowledge about melanin processing within keratinocytes. Finally, we pinpoint new questions that ought to be addressed to solve the long-lasting quest for the understanding of how basal skin pigmentation is controlled. This knowledge will allow the emergence of new strategies to treat pigmentary disorders that cause a significant socio-economic burden to patients and healthcare systems worldwide and could also have relevant cosmetic applications.publishersversionpublishe

Subversion of Ras Small GTPases in Cutaneous Melanoma Aggressiveness

The rising incidence and mortality rate associated with the metastatic ability of cutaneous melanoma represent a major public health concern. Cutaneous melanoma is one of the most invasive human cancers, but the molecular mechanisms are poorly understood. Moreover, currently available therapies are not efficient in avoiding melanoma lethality. In this context, new biomarkers of prognosis, metastasis, and response to therapy are necessary to better predict the disease outcome. Additionally, the knowledge about the molecular alterations and dysregulated pathways involved in melanoma metastasis may provide new therapeutic targets. Members of the Ras superfamily of small GTPases regulate various essential cellular activities, from signaling to membrane traffic and cytoskeleton dynamics. Therefore, it is not surprising that they are differentially expressed, and their functions subverted in several types of cancer, including melanoma. Indeed, Ras small GTPases were found to regulate melanoma progression and invasion. Hence, a better understanding of the mechanisms regulated by Ras small GTPases that are involved in melanoma tumorigenesis and progression may provide new therapeutic strategies to block these processes. Here, we review the current knowledge on the role of Ras small GTPases in melanoma aggressiveness and the molecular mechanisms involved. Furthermore, we summarize the known involvement of these proteins in melanoma metastasis and how these players influence the response to therapy.publishersversionpublishe

Commensal-to-pathogen transition: One-single transposon insertion results in two pathoadaptive traits in Escherichia coli -macrophage interaction

There are no funders and sponsors indicated explicitly in the document. This deposit is composed by the main article plus the supplementary materials of the publication.Escherichia coli is both a harmless commensal in the intestines of many mammals, as well as a dangerous pathogen. The evolutionary paths taken by strains of this species in the commensal-to-pathogen transition are complex and can involve changes both in the core genome, as well in the pan-genome. One way to understand the likely paths that a commensal strain of E. coli takes when evolving pathogenicity is through experimentally evolving the strain under the selective pressures that it will have to withstand as a pathogen. Here, we report that a commensal strain, under continuous pressure from macrophages, recurrently acquired a transposable element insertion, which resulted in two key phenotypic changes: increased intracellular survival, through the delay of phagosome maturation and increased ability to escape macrophages. We further show that the acquisition of the pathoadaptive traits was accompanied by small but significant changes in the transcriptome of macrophages upon infection. These results show that under constant pressures from a key component of the host immune system, namely macrophage phagocytosis, commensal E. coli rapidly acquires pathoadaptive mutations that cause transcriptome changes associated to the host-microbe duet.There are no funders and sponsors indicated explicitly in the document.info:eu-repo/semantics/publishedVersio

Unraveling the relevance of arl gtpases in cutaneous melanoma prognosis through integrated bioinformatics analysis

Funding Information: Funding: This research was funded by iNOVA4Health—UIDB/04462/2020, a program financially supported by the Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência. M.P. was funded by the Liga Portuguesa Contra o Cancro ‒ Núcleo Regional do Sul (LPCC‐NRS). D.B. was funded by the FCT Investigator Program (IF/00501/2014/CP1252/CT0001). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Cutaneous melanoma (CM) is the deadliest skin cancer, whose molecular pathways underlying its malignancy remain unclear. Therefore, new information to guide evidence‐based clinical decisions is required. Adenosine diphosphate (ADP)‐ribosylation factor‐like (ARL) proteins are membrane trafficking regulators whose biological relevance in CM is undetermined. Here, we investigated ARL expression and its impact on CM prognosis and immune microenvironment through integrated bioinformatics analysis. Our study found that all 22 ARLs are differentially expressed in CM. Specifically, ARL1 and ARL11 are upregulated and ARL15 is downregulated regardless of mutational frequency or copy number variations. According to TCGA data, ARL1 and ARL15 represent independent prognostic factors in CM as well as ARL11 based on GEPIA and OncoLnc. To investigate the mechanisms by which ARL1 and ARL11 increase patient survival while ARL15 reduces it, we evaluated their correlation with the immune microenvironment. CD4+ T cells and neutrophil infiltrates are significantly increased by ARL1 expression. Furthermore, ARL11 expression was correlated with 17 out of 21 immune infiltrates, including CD8+ T cells and M2 macrophages, described as having anti‐tumoral activity. Likewise, ARL11 is interconnected with ZAP70, ADAM17, and P2RX7, which are implicated in immune cell activation. Collectively, this study provides the first evidence that ARL1, ARL11, and ARL15 may influence CM progression, prognosis, and immune microenvironment remodeling.publishersversionpublishe

Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB

Funding: This study was supported by Fundaçã o para a Ciência e Tecnologia (FCT): C.E. was supported by a post-doctoral fellowship (SFRH/BPD/78491/2011), L.B.-L. by a PhD fellowship (SFRH/BD/131938/2017) and D.C.B. by the FCT Investigator Program (IF/00501/2014/CP1252/CT0001). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund). This article was supported by the LYSOCIL project. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 811087. Deposited in PMC for immediate release.Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) and also Rab3a, which we described previously as a regulator of the positioning and exocytosis of lysosomes. Thus, our study identifies new players required for lysosome exocytosis and suggest the existence of a Rab11-Rab3a cascade involved in this process.publishersversionpublishe

Reconstructed human pigmented skin/epidermis models achieve epidermal pigmentation through melanocore transfer

Funding: We thank the Electron Microscopy Facility at the Instituto Gulbenkian de Ciência, the Pathological Anatomy service in the Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG) and the Histology Facility at CEDOC for technical assistance. This project was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal through FCT Unit iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a programme financially supported by FCT / Ministério da Ciência, Tecnologia e Ensino Superior, through national funds, grant PTDC/BIA-CEL/29765/2017, PhD fellowships 2020.08528.BD (SLV), PD/BD/128164/2016, PD/BD/137442/2018 (MVN) and PD/BD/136905/2018 (JC) and the FCT Investigator Program to DCB (IF/00501/2014/CP1252/CT0001).The skin acts as a barrier to environmental insults and provides many vital functions. One of these is to shield DNA from harmful UV radiation, which is achieved by skin pigmentation arising as melanin is produced and dispersed within the epidermal layer. This is a crucial defence against DNA damage, photo-ageing and skin cancer. The mechanisms and regulation of melanogenesis and melanin transfer involve extensive crosstalk between melanocytes and keratinocytes in the epidermis, as well as fibroblasts in the dermal layer. Although the predominant mechanism of melanin transfer continues to be debated and several plausible models have been proposed, we and others previously provided evidence for a coupled exo/phagocytosis model. Herein, we performed histology and immunohistochemistry analyses and demonstrated that a newly developed full-thickness 3D reconstructed human pigmented skin model and an epidermis-only model exhibit dispersed pigment throughout keratinocytes in the epidermis. Transmission electron microscopy revealed melanocores between melanocytes and keratinocytes, suggesting that melanin is transferred through coupled exocytosis/phagocytosis of the melanosome core, or melanocore, similar to our previous observations in human skin biopsies. We therefore present evidence that our in vitro models of pigmented human skin show epidermal pigmentation comparable to human skin. These findings have a high value for studies of skin pigmentation mechanisms and pigmentary disorders, whilst reducing the reliance on animal models and human skin biopsies.publishersversionepub_ahead_of_prin

RAB3A Regulates Melanin Exocytosis and Transfer Induced by Keratinocyte-Conditioned Medium

Funding: We would like to thank our group for the critical reading of the manuscript and the NMS microscopy and cell culture facilities, as well as José Belo’s group, for the kind gift of mouse embryonic fibroblasts. This study was supported by Fundação para a Ciência e a Tecnologia (Portugal) through grant PTDC/BIA-CEL/29765/2017 and PhD fellowships to LCC, MVN, LBL and AF (2020.08812.BD, PD/BD/137442/2018, SFRH/BD/131938/2017 and PD/BD/ 135506/2018, respectively). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, cofinanced by Fundação para a Cieˆncia e a Tecnologia (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund). This article was supported by the LYSOCIL project, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 811087. This work was also supported by iNOVA4Health e UIDB/04462/2020 and UIDP/04462/2020 and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), two programs financially supported by Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (Portugal).Skin pigmentation is imparted by melanin and is crucial for photoprotection against UVR. Melanin is synthesized and packaged into melanosomes within melanocytes and is then transferred to keratinocytes (KCs). Although the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer remain unclear. Previously, our group proposed that coupled exocytosis/phagocytosis is the predominant mechanism of melanin transfer in human skin and showed an essential role for RAB11B and the exocyst tethering complex in this process. In this study, we show that soluble factors present in KC-conditioned medium stimulate melanin exocytosis from melanocytes and transfer to KCs. Moreover, we found that these factors are released by differentiated KCs but not by basal layer KCs. Furthermore, we found that RAB3A regulates melanin exocytosis and transfer stimulated by KC-conditioned medium. Indeed, KC-conditioned medium enhances the recruitment of RAB3A to melanosomes in melanocyte dendrites. Therefore, our results suggest the existence of two distinct routes of melanin exocytosis: a basal route controlled by RAB11B and a RAB3A-dependent route, stimulated by KC-conditioned medium. Thus, this study provides evidence that soluble factors released by differentiated KCs control skin pigmentation by promoting the accumulation of RAB3A-positive melanosomes in melanocyte dendrites and their release and subsequent transfer to KCs.publishersversionpublishe

Melanin transferred to keratinocytes resides in non-degradative endocytic compartments

Funding: We thank the staff from the Unit of Imaging and Cytometry of the Instituto Gulbenkian de Ciência for assistance in microscopy and flow cytometry protocol adjustments, the CEDOC Microscopy Facility for assistance in image acquisition and analysis, Dot Bennett for the kind gift of cell lines, and Alistair Hume for the kind gift of anti-Rab5 antibody. This project was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (PTDC/BIA-BCM/ 111735/2009, EXPL/BEX-BCM/0379/2013), MSC and HM were supported by FCT PhD studentships (SFRH/BD/65381/2009 and PD/BD/114118/2015, respectively), FJCP was supported by an FCT postdoctoral fellowship (SFRH/ BPD/70337/2010), and DCB was supported by the FCT Investigator Program (IF/00501/2014/CP1252/CT0001).Melanin transfer from melanocytes to keratinocytes and subsequent accumulation in the supra-nuclear region is a critical process in skin pigmentation and protection against ultraviolet radiation. We have previously proposed that the main mode of transfer between melanocytes and keratinocytes is through exo/endocytosis of the melanosome core, termed melanocore. In this study, we developed an in vitro uptake assay using melanocores secreted by melanocytes. We show that the uptake of melanocores, but not melanosomes, by keratinocytes is Protease-activated receptor (PAR)-2-dependent. Furthermore, we found that the silencing of the early endocytic regulator Rab5b, but not the late endocytic regulators Rab7a or Rab9a, significantly impairs melanocore uptake by keratinocytes. Following uptake, we observed that melanin accumulates in compartments that are positive for both early and late endocytic markers. We found that melanin does not localize to either highly degradative or acidic organelles, as assessed by LysoTracker and DQ-BSA staining, despite the abundance of these types of organelles within keratinocytes. Therefore, we propose that melanocore uptake leads to storage of melanin within keratinocytes in hybrid endocytic compartments that are not highly acidic or degradative. By avoiding lysosomal degradation, these specialized endosomes may allow melanin to persist within keratinocytes for long periods.publishersversionpublishe

Regulation of CD1 Antigen-presenting Complex Stability

For major histocompatibility complex class I and II molecules, the binding of specific peptide antigens is essential for assembly and trafficking and is at the center of their quality control mechanism. However, the role of lipid antigen binding in stabilization and quality control of CD1 heavy chain (HC).beta(2)-microglobulin (beta(2)m) complexes is unclear. Furthermore, the distinct trafficking and loading routes of CD1 proteins take them from mildly acidic pH in early endososmal compartments (pH 6.0) to markedly acidic pH in lysosomes (pH 5.0) and back to neutral pH of the cell surface (pH 7.4). Here, we present evidence that the stability of each CD1 HC.beta(2)m complex is determined by the distinct pH optima identical to that of the intracellular compartments in which each CD1 isoform resides. Although stable at acidic endosomal pH, complexes are only stable at cell surface pH 7.4 when bound to specific lipid antigens. The proposed model outlines a quality control program that allows lipid exchange at low endosomal pH without dissociation of the CD1 HC.beta(2)m complex and then stabilizes the antigen-loaded complex at neutral pH at the cell surface