Spatiotemporal metabolic dynamics of the photosensitizer talaporfin sodium in carcinoma and sarcoma

Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co‐localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin‐ and caveolae‐dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K‐Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin‐ and caveolae‐dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K‐Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma.     

The Salmonella virulence protein SifA is a G protein antagonist

Salmonella's success at proliferating intracellularly and causing disease depends on the translocation of a major virulence protein, SifA, into the host cell. SifA recruits membranes enriched in lysosome associated membrane protein 1 (LAMP1) and is needed for growth of Salmonella induced filaments (Sifs) and the Salmonella containing vacuole (SCV). It directly binds a host protein called SKIP (SifA and kinesin interacting protein) which is critical for membrane stability and motor dynamics at the SCV. SifA also contains a WxxxE motif, predictive of G protein mimicry in bacterial effectors, but whether and how it mimics the action of a host G protein is not known. We show that SKIP's pleckstrin homology domain, which directly binds SifA, also binds to the late endosomal GTPase Rab9. Knockdown studies suggest that both SKIP and Rab9 function to maintain peripheral LAMP1 distribution in cells. The Rab9:SKIP interaction is GTP-dependent and is inhibited by SifA binding to the SKIP pleckstrin homology domain, suggesting that SifA may be a Rab9 antagonist. SifA:SKIP binding is significantly tighter than Rab9:SKIP binding and may thus allow SifA to bring SKIP to the SCV via SKIP's Rab9-binding site. Rab9 can measurably reverse SifA-dependent LAMP1 recruitment and the perinuclear location of the SCV in cells. Importantly, binding to SKIP requires SifA residues W197 and E201 of the conserved WxxxE signature sequence, leading to the speculation that bacterial G protein mimicry may result in G protein antagonism.     

Autophagy is required for proper cysteine homeostasis in pancreatic cancer through regulation of SLC7A11

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.

Despite progress in cancer therapy, the prognosis for pancreatic ductal adenocarcinoma (PDAC) remains extremely poor with a 5-y survival rate of just 9% and it is predicted to become the second leading cause of cancer death in the United States by 2030 (1–3). The PDAC tumor microenvironment is highly desmoplastic and is composed of heterogeneous cell types, as well as an exuberant extracellular matrix. Together, this leads to poor perfusion and extreme hypoxia, creating a nutrient-limited environment with impaired drug penetration (4, 5). Another hallmark of PDAC is elevated basal autophagy which plays multiple protumorigenic roles, including promoting immune evasion and supporting its metabolic demand in this austere microenvironment (6–10). Therefore, clinical strategies have been employed to inhibit autophagy in PDAC patients using lysosomal inhibitors such as chloroquine or hydroxychloroquine (11, 12).While autophagy can support diverse metabolic processes through the degradation of various cargo, how it supports PDAC metabolism has not been fully elucidated. In the present study, we found that autophagy has a selective role in sustaining cysteine (Cys) pools in PDAC. One of the major mechanisms of Cys homeostasis is through the import of cystine (the oxidized dimer of cysteine) through system x_c⁻, a cystine/glutamate antiporter composed of SLC7A11 (xCT) and SLC3A2 (13). Recently, it was shown that both Cys and SLC7A11 are critical for PDAC growth (14). Here, we report that under low Cys conditions, SLC7A11 utilizes autophagy machinery to allow localization at the plasma membrane. Moreover, we demonstrate that loss of autophagy increases phosphorylation of SLC7A11 by mTORC2, and it remains primarily localized at the lysosome where its cystine import function is impaired. In summary, we identify a mechanism of Cys homeostasis in PDAC where the function of SLC7A11 is coordinately sustained by autophagic machinery and mTORC2 activity based on intracellular Cys levels.     

A lipid switch unlocks Parkinson’s disease-associated ATP13A2

ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn²⁺, and Zn²⁺ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders.Neuronal fitness depends on optimal lysosomal function and efficient lysosomal delivery of proteins and organelles by autophagy for subsequent breakdown (1, 2). Kufor–Rakeb syndrome (KRS) is an autosomal recessive form of Parkinson’s disease (PD) associated with dementia, which is caused by mutations in ATP13A2/PARK9 (3). Mutations in or knockdown (KD) of ATP13A2 lead to lysosomal dysfunctions, including reduced lysosomal acidification, decreased degradation of lysosomal substrates (4), impaired autophagosomal flux (4, 5), and accumulation of fragmented mitochondria (5, 6). By contrast, overexpression (OE) of Ypk9p (i.e., the yeast ATP13A2 ortholog) protects yeast against toxicity of α-synuclein (7), which is the major protein in Lewy bodies, the abnormal protein aggregates that develop inside nerve cells in PD. This protective effect of ATP13A2 on α-synuclein toxicity is conserved in yeast, Caenorhabditis elegans, and rat neuronal cells (7). Because ATP13A2 imparts resistance to Mn²⁺ (7–9) and Zn²⁺ (10–12), it was proposed that ATP13A2 may function as a Mn²⁺ (7–9) and/or Zn²⁺ transporter (10–12).ATP13A2 belongs to the P5 subfamily of the P-type ATPase superfamily, which comprises five subfamilies (P1–5) of membrane transporters. P-type ATPases hydrolyze ATP to actively transport inorganic ions across membranes or lipids between membrane leaflets (reviewed in ref. 13). During the transport cycle, a phospho-intermediate is formed on a conserved aspartate residue (14). The human P5-type ATPases are divided into two groups, P5A (ATP13A1) and P5B (ATP13A2–5), but their transport specificity has not been established (14–16).P-type ATPases comprise a membrane-embedded core of six transmembrane (TM) helices (M1–6) that form the substrate binding site(s) and entrance/exit pathways for the transported substrate (13). Whereas four extra C-terminal TM helices (M7–10) are common, additional N-terminal TM helices are only observed in the P1B heavy metal pumps (17). Topology predictions indicate that ATP13A2 and other P5 members also contain additional N-terminal TM helices, which might serve a subclass-specific function (14, 15). Here, we demonstrate that the ATP13A2 N terminus is a critical regulatory element involved in lipid binding and ATP13A2 activation, providing protection to mitochondrial stress in a cellular PD model.     

抗疟药物的心脏毒性

虽然长期应用抗疟药物诱发心脏毒性者罕见,但后果严重。临床主要表现为心脏传导阻滞、充血性心力衰竭、心律失常及心肌病。确诊需行心肌心内膜活检。光学显微镜下组织病理学改变为心肌细胞内大量空泡形成,电子显微镜下见髓样体及特征性的曲线体。抗疟药物被溶酶体选择性摄取后使溶酶体pH值升高,从而干扰溶酶体中酶类活性,导致糖原及磷脂积聚。     

Autophagy as an antimicrobial strategy

Autophagy is a process of lysosomal degradation that was originally described as a cellular response to adapt to a lack of nutrients and to enable the elimination of damaged organelles. Autophagy is increasingly recognized as a process that is also involved in innate and adaptive immune responses against pathogens. Studies on the regulation of autophagy have uncovered components of the autophagic cascade that can be manipulated pharmacologically. Approaches to modulate autophagy may result in novel strategies for the treatment and prevention of various infections.     

Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.Autophagy is the major lysosome/vacuole-dependent cellular degradative pathway. During autophagy, cytoplasmic constituents including proteins, lipids, and even entire organelles are surrounded by the phagophore, the initial sequestering compartment. The phagophore then expands to form double-membrane vesicles, termed autophagosomes. The completed autophagosomes fuse with lysosomes/vacuoles allowing access of the cargo to the degradative enzymes within this organelle; the resulting breakdown products are released back into the cytosol as building blocks or catabolic substrates (1–3). Autophagy is not only critical for survival during nutrient deprivation but is also involved in various human pathophysiologies, including cancer and neurodegeneration (4).On autophagy induction, AuTophaGy-related (Atg) proteins accumulate at the phagophore assembly site (PAS) and initiate autophagosome formation. Among the 36 known Atg proteins, Atg1 is the only kinase, and it plays a particularly important role in autophagy induction by controlling the movement of other Atg proteins including Atg9 and Atg23 (5) and in the proper organization of the PAS (6, 7). Atg1 interacts with several proteins, including direct binding to Atg13 (which interacts with Atg17) and Atg11; the kinase activity of Atg1 is regulated in part by its binding to, and/or interaction with, some of these components (8, 9).Atg17 constitutively forms a stable protein complex with Atg29 and Atg31 in both growing and nitrogen starvation conditions (10), and Atg31 directly interacts with Atg17 and Atg29 to bridge these two proteins (11). When autophagy is initiated, the Atg17-Atg31-Atg29 complex is first targeted to the PAS and recruits other Atg proteins, including Atg1 and Atg13, highlighting the significance of the ternary complex (12, 13). Along these lines, a single deletion of the ATG17, ATG29, or ATG31 genes results in a dramatic decrease in autophagy activity (14–16).In this study, we examined the role of posttranslational modification of Atg29. We found that Atg29 is a phosphoprotein and that phosphorylation on the C-terminal domain is critical for autophagy activity; the N terminus of Atg29 contains the functional domain, whereas the C terminus plays a regulatory role. We continued and extended our study to include a structural and functional analysis of the Atg17-Atg31-Atg29 complex. Single-particle electron microscopy (EM) reveals that the recombinant Atg17-Atg31-Atg29 complex is present as an elongated S-shaped dimerized structure, with Atg17 forming the backbone. We further demonstrate that Atg29 has the characteristics of an intrinsically disordered protein (IDP), suggesting that the C-terminal half is flexible and capable of altering its conformation on binding to one or more interacting proteins. Finally, we determined that Atg11 is necessary and sufficient to recruit this complex to the PAS and that phosphorylation of Atg29 is required for its interaction with Atg11 and proper PAS localization.     

Lack of correlation between lysosomal enzyme activity and ascorbic acid content in brain of lead-treated animals

The administration of lead acetate increased acid phosphatase activities in the rat brain and four regions (cerebral cortex, diencephalon plus mesencephalon, pons plus medulla, and cerebellum) of the guinea pig brain, whereas ascorbic acid content in the brain of these animals remained unchanged following lead administration. Acid phosphatase activity also showed an increase in scorbutic guinea pig brain. Lipid peroxidation facilitated by ascorbic acid in the cerebral lysosome fraction of rat was not affected by lead. These results suggest that ascorbic acid is not directly concerned with the alteration in acid phosphatase activity induced by lead treatment.