GRASPing the unconventional secretory machinery to bridge cellular stress signaling to the extracellular proteome

Cellular adaptation to stress is a crucial homeostatic process for survival, metabolism, physiology, and disease. Cells respond to stress stimuli (e.g., nutrient starvation, growth factor deprivation, hypoxia, low energy, etc.) by changing the activity of signaling pathways, and interact with their environment by qualitatively and quantitatively modifying their intracellular, surface, and extracellular proteomes. How this delicate communication takes place is a hot topic in cell biological research, and has important implications for human disease.

Translation Inhibitors Activate Autophagy Master Regulators TFEB and TFE3

The autophagy-lysosome pathway is a major protein degradation pathway stimulated by multiple cellular stresses, including nutrient or growth factor deprivation, hypoxia, misfolded proteins, damaged organelles, and intracellular pathogens. Recent studies have revealed that transcription factor EB (TFEB) and transcription factor E3 (TFE3) play a pivotal role in the biogenesis and functions of autophagosome and lysosome. Here we report that three translation inhibitors (cycloheximide, lactimidomycin, and rocaglamide A) can facilitate the nuclear translocation of TFEB/TFE3 via dephosphorylation and 14-3-3 dissociation. In addition, the inhibitor-mediated TFEB/TFE3 nuclear translocation significantly increases the transcriptional expression of their downstream genes involved in the biogenesis and function of autophagosome and lysosome. Furthermore, we demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. These results highlight the previously unrecognized function of the translation inhibitors as activators of TFEB/TFE3, suggesting a novel biological role of translation inhibition in autophagy regulation.

mTOR repression in response to amino acid starvation promotes ECM degradation through MT1-MMP endocytosis arrest

Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism towards the consumption of extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here we show that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, we found that collagenolysis requires the invadopodia components, TKS5 and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. Our results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. Our study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies.

Class I PI3K Is Required for HSC Differentiation

Adult hematopoietic stem cells (HSCs) are a rare and unique population of stem cells that reside in the bone marrow, where they undergo self-renewal and differentiation to maintain the blood system. The maintenance of a proper balance between HSC self-renewal and differentiation requires growth factors, cytokines, and chemokines, most of which activate the phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. Pathologic activation of the AKT pathway is frequently observed in tumors, making it a desirable target for cancer treatment. Since several PI3K inhibitors are now in clinical use, it is critical to determine the roles of PI3K in adult HSCs. However, the specific roles of PI3K in HSC function are poorly understood. Hematopoietic cells express three Class IA catalytic PI3K isoforms (P110α, β, and δ), which can all transduce growth factor and cytokine signals, and can compensate for one another in some cell types. Individual Class 1A PI3K isoforms have unique functions in mature hematopoietic lineages, but are dispensable for HSC function. To uncover the potentially redundant roles of PI3K isoforms in HSCs, we have generated a triple knockout (TKO) mouse model with conditional deletion of p110α and p110β in hematopoietic cells using MX1-Cre, and germline deletion of p110δ. TKO mice develop pancytopenia, which is also observed upon transplantation of TKO bone marrow. Competitive repopulation assays reveal a defect in long-term multi-lineage chimerism. Surprisingly, loss of Class 1A PI3K causes significant expansion of donor-derived long-term (Lin-cKit+Flk2-CD150+CD48-) and short-term (Lin-cKit+Flk2-CD150-CD48-) HSCs in the bone marrow, but not committed progenitors. This phenotype could not be explained by alterations in HSC cell cycling or apoptosis in TKO HSCs. TKO transplant recipients also have dysplastic features in the bone marrow. Methylcellulose plating assays of TKO bone marrow revealed a relative increase in granulocyte erythroid macrophage megakaryocyte (GEMM) colonies and extended serial replating, suggesting increased self-renewal. Thus, our data are consistent with impaired HSC differentiation upon deletion of all Class IA PI3K isoforms, which leads to dysplastic changes. RNA sequencing of sorted long-term HSCs from the bone marrow of TKO transplant recipients revealed the enrichment of human and mouse HSC signatures, and the downregulation of DNA repair gene sets and RNA splicing gene sets in TKO HSCs. Interestingly, we also observed downregulation of autophagy gene sets in TKO HSCs. Macroautophagy has been shown to be essential for the maintenance of HSC metabolism and self-renewal. Analysis of the autophagosomal marker LC3-II in TKO HSCs revealed a decrease in autophagy upon growth factor deprivation. Surprisingly, we observed an increase in MTOR activation in TKO cKit+ bone marrow cells via compensatory signaling through the MAPK pathway. Given that MTOR is a known negative regulator of autophagy, this is consistent with the observed autophagy decrease in TKO HSCs. Additionally, we found that autophagy can still be induced in TKO HSCs with the MTOR inhibitor rapamycin. Furthermore, rapamycin treatment impairs serial replating of TKO bone marrow cells. In conclusion, we found that inactivation of all Class 1A PI3 kinases leads to impaired HSC differentiation, likely due to a defect in autophagy induction in response to growth factor deprivation. Disclosures No relevant conflicts of interest to declare.

Host–pathogen interactions and subversion of autophagy

Macroautophagy (‘autophagy’), is the process by which cells can form a double-membraned vesicle that encapsulates material to be degraded by the lysosome. This can include complex structures such as damaged mitochondria, peroxisomes, protein aggregates and large swathes of cytoplasm that can not be processed efficiently by other means of degradation. Recycling of amino acids and lipids through autophagy allows the cell to form intracellular pools that aid survival during periods of stress, including growth factor deprivation, amino acid starvation or a depleted oxygen supply. One of the major functions of autophagy that has emerged over the last decade is its importance as a safeguard against infection. The ability of autophagy to selectively target intracellular pathogens for destruction is now regarded as a key aspect of the innate immune response. However, pathogens have evolved mechanisms to either evade or reconfigure the autophagy pathway for their own survival. Understanding how pathogens interact with and manipulate the host autophagy pathway will hopefully provide a basis for combating infection and increase our understanding of the role and regulation of autophagy. Herein, we will discuss how the host cell can identify and target invading pathogens and how pathogens have adapted in order to evade destruction by the host cell. In particular, we will focus on interactions between the mammalian autophagy gene 8 (ATG8) proteins and the host and pathogen effector proteins.

Receptor tyrosine kinase Met promotes cell survival via kinase-independent maintenance of integrin α3β1

Matrix adhesion via integrins is required for cell survival. Adhesion of epithelial cells to laminin via integrin α3β1 was previously shown to activate at least two independent survival pathways. First, integrin α3β1 is required for autophagy-induced cell survival after growth factor deprivation. Second, integrin α3β1 independently activates two receptor tyrosine kinases, EGFR and Met, in the absence of ligands. EGFR signaling to Erk promotes survival independently of autophagy. To determine how Met promotes cell survival, we inhibited Met kinase activity or blocked its expression with RNA interference. Loss of Met expression, but not inhibition of Met kinase activity, induced apoptosis by reducing integrin α3β1 levels, activating anoikis, and blocking autophagy. Met was specifically required for the assembly of autophagosomes downstream of LC3II processing. Reexpression of wild-type Met, kinase-dead Met, or integrin α3 was sufficient to rescue death upon removal of endogenous Met. Integrin α3β1 coprecipitated and colocalized with Met in cells. The extracellular and transmembrane domain of Met was required to fully rescue cell death and restore integrin α3 expression. Thus Met promotes survival of laminin-adherent cells by maintaining integrin α3β1 via a kinase-independent mechanism.

Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status

Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows.