bims-momema Biomed News
on Molecular mechanisms of macropinocytosis
Issue of 2022‒04‒24
seven papers selected by
Harilaos Filippakis
Harvard University

  1. Pharmaceutics. 2022 Apr 13. pii: 855. [Epub ahead of print]14(4):
      The iron-binding protein lactoferrin and the cell-penetrating peptides derived from its sequence utilise endocytosis to enter different cell types. The full-length protein has been extensively investigated as a potential therapeutic against a range of pathogenic bacteria, fungi, and viruses, including SARS-CoV-2. As a respiratory antiviral agent, several activity mechanisms have been demonstrated for lactoferrin, at the extracellular and plasma membrane levels, but as a protein that enters cells it may also have intracellular antiviral activity. Characterisation of lactoferrin's binding, endocytic traffic to lysosomes, or recycling endosomes for exocytosis is lacking, especially in lung cell models. Here, we use confocal microscopy, flow cytometry, and degradation assays to evaluate binding, internalisation, endocytic trafficking, and the intracellular fate of bovine lactoferrin in human lung A549 cells. In comparative studies with endocytic probes transferrin and dextran, we show that lactoferrin binds to negative charges on the cell surface and actively enters cells via fluid-phase endocytosis, in a receptor-independent manner. Once inside the cell, we show that it is trafficked to lysosomes where it undergoes degradation within two hours. These findings provide opportunities for investigating both lactoferrin and derived cell-penetrating peptides activities of targeting intracellular pathogens.
    Keywords:  endocytosis; intracellular trafficking; lactoferrin; lysosomal degradation
  2. Membranes (Basel). 2022 Apr 01. pii: 393. [Epub ahead of print]12(4):
      Endocytosis is a critical process for cell growth and viability. It mediates nutrient uptake, guarantees plasma membrane homeostasis, and generates intracellular signaling cascades. Moreover, it plays an important role in dead cell clearance and defense against external microbes. Finally, endocytosis is an important cellular route for the delivery of nanomedicines for therapeutic treatments. Thus, it is not surprising that both environmental and genetic perturbation of endocytosis have been associated with several human conditions such as cancer, neurological disorders, and virus infections, among others. Over the last decades, a lot of research has been focused on developing advanced imaging methods to monitor endocytosis events with high resolution in living cells and tissues. These include fluorescence imaging, electron microscopy, and correlative and super-resolution microscopy. In this review, we outline the major endocytic pathways and briefly discuss how defects in the molecular machinery of these pathways lead to disease. We then discuss the current imaging methodologies used to study endocytosis in different contexts, highlighting strengths and weaknesses.
    Keywords:  correlative microscopy; disease; electron microscopy; endocytosis; fluorescence; protein receptor; super-resolution microscopy
  3. Mol Plant Pathol. 2022 Apr 19.
      All organisms need to sense and process information about the availability of nutrients, energy status, and environmental cues to determine the best time for growth and development. The conserved target of rapamycin (TOR) protein kinase has a central role in sensing and perceiving nutritional information. TOR connects environmental information about nutrient availability to developmental and metabolic processes to maintain cellular homeostasis. Under favourable energy conditions, TOR is activated and promotes anabolic processes such as cell division, while suppressing catabolic processes. Conversely, when nutrients are limited or environmental stresses are present, TOR is inactivated, and catabolic processes are promoted. Given the central role of TOR in regulating metabolism, several previous works have examined whether TOR is wired to plant defence. To date, the mechanisms by which TOR influences plant defence are not entirely clear. Here, we addressed this question by testing the effect of inhibiting TOR on immunity and pathogen resistance in tomato. Examining which hormonal defence pathways are influenced by TOR, we show that tomato immune responses and disease resistance to several pathogens increase on TOR inhibition, and that TOR inhibition-mediated resistance probably requires a functional salicylic acid, but not jasmonic acid, pathway. Our results support the notion that TOR is a master regulator of the development-defence switch in plants.
    Keywords:   Botrytis ; Xanthomonas ; TOR; immunity; pathogenesis; tobacco mosaic virus; tomato
  4. Transl Oncol. 2022 Apr 13. pii: S1936-5233(22)00081-X. [Epub ahead of print]20 101421
      Non-small cell lung cancer (NSCLC) ranks first among cancer death worldwide. Despite efficacy and safety priority, targeted therapy only benefits ∼30% patients, leading to the unchanged survival rates for whole NSCLC patients. Metabolic reprogramming occurs to offer energy and intermediates for fuelling cancer cells proliferation. Thus, mechanistic insights into metabolic reprogramming may shed light upon NSCLC proliferation and find new proper targets for NSCLC treatment. Herein, we used loss- and gain-of-function experiments to uncover that highly expressed aldo-keto reductase family1 member C1 (AKR1C1) accelerated NSCLC cells proliferation via metabolic reprogramming. Further molecular profiling analyses demonstrated that AKR1C1 augmented the expression of hypoxia-inducible factor 1-alpha (HIF-1α), which could drive tumour metabolic reprogramming. What's more, AKR1C1 significantly correlated with HIF-1α signaling, which predicted poor prognosis for NSCLC patients. Collectively, our data display that AKR1C1 reprograms tumour metabolism to promote NSCLC cells proliferation by activating HIF-1α. These newly acquired data not only establish the specific role for AKR1C1 in metabolic reprogramming, but also hint to the possibility that AKR1C1 may be a new therapeutic target for NSCLC treatment.
    Keywords:  AKR1C1; HIF-1α; Metabolic reprogramming; Proliferation
  5. Biomolecules. 2022 Apr 08. pii: 553. [Epub ahead of print]12(4):
      Bronchial asthma is a chronic disease characterized by airway inflammation, obstruction, and hyperresponsiveness. CD4+ T cells, particularly T helper (Th) 2 cells, and their specific cytokines are important mediators in asthma pathogenesis. However, it has been established that Th subsets, other than Th2, as well as various cell types, including innate lymphoid cells (ILCs), significantly contribute to the development of allergic inflammation. These cells require facilitated amino acid uptake to ensure their full function upon activation. Emerging studies have suggested the potential of pharmacological inhibition of amino acid transporters to inhibit T cell activation and the application of this strategy for treating immunological and inflammatory disorders. In the present review, we explore the possibility of targeting L-type amino acid transporter (LAT) as a novel therapeutic approach for bronchial asthma, including its steroid-resistant endotypes.
    Keywords:  L-type amino acid transporter (LAT) 1; Th17; steroid-resistant asthma
  6. Autophagy Rep. 2022 ;1(1): 88-118
      Autophagosome formation involves the sequential actions of conserved ATG proteins to coordinate the lipidation of the ubiquitin-like modifier Atg8-family proteins at the nascent phagophore membrane. Although the molecular steps driving this process are well understood, the source of membranes for the expanding phagophore and their mode of delivery are only now beginning to be revealed. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12-ATG5 conjugate, a crucial player during Atg8-family protein lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins alters autophagic flux in cell-based starvation assays. To determine whether the ATG12-ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. We report changes in the relative proportions of individual cell surface proteins and show that cell surface levels of the SLC7A5-SLC3A2 amino acid transporter are influenced by autophagy capability. Our data provide evidence for direct regulatory coupling between the ATG12-ATG5 conjugate and the clathrin membrane trafficking system and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery. Abbreviations: ATG, autophagy related; BafA1, bafilomycin A1; GFP, green fluorescent protein; HIP1R, huntingtin interacting protein 1 related; MEF, mouse embryo fibroblast; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha; SILAC, stable isotope labelling with amino acids in culture; SQSTM1, sequestosome 1; STRING, search tool for the retrieval of interacting genes/proteins.
    Keywords:  ATG12; ATG5; HIP1R; PIK3C2A; autophagy; clathrin; endocytosis; proteomics
  7. Oncogene. 2022 Apr 22.
      Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of RMS patients.