Amphibian pore-forming protein βγ-CAT drives extracellular nutrient scavenging under cell nutrient deficiency

Summary Nutrient acquisition is essential for animal cells. βγ-CAT is a pore-forming protein (PFP) and trefoil factor complex assembled under tight regulation identified in toad Bombina maxima. Here, we reported that B. maxima cells secreted βγ-CAT under glucose, glutamine, and pyruvate deficiency to scavenge extracellular proteins for their nutrient supply and survival. AMPK signaling positively regulated the expression and secretion of βγ-CAT. The PFP complex selectively bound extracellular proteins and promoted proteins uptake through endolysosomal pathways. Elevated intracellular amino acids, enhanced ATP production, and eventually prolonged cell survival were observed in the presence of βγ-CAT and extracellular proteins. Liposome assays indicated that high concentration of ATP negatively regulated the opening of βγ-CAT channels. Collectively, these results uncovered that βγ-CAT is an essential element in cell nutrient scavenging under cell nutrient deficiency by driving vesicular uptake of extracellular proteins, providing a new paradigm for PFPs in cell nutrient acquisition and metabolic flexibility.

The secreted bg-CAT selectively binds and scavenges extracellular nutrients The high concentrations of ATP may negatively regulate bg-CAT channel opening

INTRODUCTION
In animal cells, plasma membrane transporters and receptor-mediated endocytosis of nutrient carriers are the two main canonical ways by which cells import nutrients for their life cycle. The former operates in uptake of small nutrient, such as glucose and amino acids, and the latter is the main pathway through which cells obtain insoluble nutrients, such as cholesterol and iron. 1,2 Additionally, animal cells ingest extracellular macromolecules through endolysosomal systems via pinocytosis, an evolutionarily conserved form of endocytosis mediating non-selective import of fluid and solutes contained therein. [3][4][5] Animal cells import macromolecules such as proteins and degrade them in lysosomes to support their metabolism and growth, such as in cancer cell metabolism. 5,6 The regulation and coordination of distinct nutrient acquisition strategies are incompletely understood. 2,7,8 The possible existence and identity of extracellular elements dispatched by cells for nutrient sampling and scavenging under nutrient deficiency require exploration.
Numerous pore-forming proteins (PFPs) with a membrane insertion domain similar to bacterial toxin aerolysin, namely aerolysin family PFPs (af-PFPs, previously referred to as aerolysin-like proteins, ALPs), have been found in plants and animals. [9][10][11] BmALP1 is an af-PFP from toad Bombina maxima. It forms membrane pores (channels) with a functional diameter 1.5-2.0 nm. [12][13][14] This PFP is regulated by environmental cues and interacts with a trefoil factor (BmTFF3) to form a PFP complex bg-CAT, in which BmTFF3 acts as a chaperon and a regulatory unit of BmALP1. [14][15][16][17] BmALP3, a paralog of BmALP1, lacks a membrane pore-forming capacity, but it oxidizes BmALP1 to its water-soluble polymer, leading to the dissociation of bg-CAT complex and loss of biological activity. 17 This PFP complex firstly targets cell surface acidic glycosphingolipids in lipid rafts via a double-receptor binding model, corresponding to BmALP1 subunit and BmTFF3 subunit binding gangliosides and sulfatides, respectively. 16 And the PFP acts along cell endocytic and exocytic pathways with channel formation on endolysosomes, which have been shown to play roles in immune defense and tissue repair. [18][19][20][21][22] Thus, the PFP and its regulatory network define an unknown secretory endolysosomal channel (SELC) pathway,

Toad cells secrete bg-CAT under nutrient deficiency
Previous studies have shown that secreted bg-CAT promotes cell import through endolysosomal pathways probably via inducing pinocytosis/macropinocytosis in distinct cell context. 13,21,24 This raises the possibility that the PFP protein complex participates in macromolecule intake for cellular nutrient supply and metabolic flexibility under variations in nutrient availability. 13,23,24 To test this hypothesis, we used a nutrient deficiency model of toad cells cultured under three nutrient conditions including glucose/glutamine/pyruvate-containing medium (Glc + /Gln + /Pyr + ), glucose/glutamine/pyruvate-depleted medium (Glcˉ/Glnˉ/Pyrˉ), and glucose-containing but glutamine/pyruvate-depleted medium (Glc + /Glnˉ/Pyrˉ). In the isolated toad liver cell population, there were 62.4% hepatocytes ( Figure S1A) as assessed by a specific antibody against hepatocyte marker cytokeratin 18 (CK18). 25,26 Under Glcˉ/Glnˉ/Pyrˉconditions, the expression of bg-CAT a-subunit BmALP1 was attenuated at 1 h, but substantially upregulated at 3 h and 5 h as analyzed by qRT-PCR ( Figure 1A) and Western blotting ( Figure 1B). Meanwhile, the expression of bg-CAT b-subunit BmTFF3 was also upregulated at 3 h ( Figure 1A). Because the PFP complex bg-CAT is a secreted protein, we next investigated the change in the bg-CAT protein level in toad liver cell supernatants by blotting for its a-subunit BmALP1. Under Glcˉ/Glnˉ/Pyrˉconditions, largely augmented secretion of bg-CAT a-subunit BmALP1 was readily detected by Western blotting ( Figure 1C). Biologically active bg-CAT in culture supernatants was analyzed by its hemolytic activity on human erythrocytes, a sensitive method to determine the existence of bg-CAT. 13,17 First, we verified that the various culture media did not affect the hemolytic activity of bg-CAT ( Figure S1B). Intriguingly, the hemolytic activity in culture supernatants of toad liver cells was largely increased under Glcˉ/Glnˉ/Pyrˉconditions compared with that of Glc + /Gln + /Pyr + conditions ( Figure 1D), which was abolished by anti-bg-CAT antibodies and anti-BmTFF3 antibodies, respectively (Figure S1C). These results indicated bg-CAT was secreted in culture supernatants of liver cells under nutrient deficiency.
Next, we investigated secretion of bg-CAT under Glc + /Glnˉ/Pyrˉconditions. The hemolytic activity assay of culture supernatants showed that the secretion of bg-CAT was substantially decreased in the liver cell supernatants under Glc + /Glnˉ/Pyrˉconditions as compared with that of Glcˉ/Glnˉ/Pyrˉconditions (Figure 1E), indicating that the presence of glucose largely decreased bg-CAT secretion in toad liver cells. A similar result was observed in toad intestinal cells ( Figure 1F). However, the secretion of bg-CAT was only partially attenuated under Glc + /Glnˉ/Pyrˉconditions in toad stomach cells under our assay conditions ( Figure 1G). These results showed that toad cells from the alimentary system secrete bg-CAT to counteract nutrient deficiency, and glutamine and pyruvate depletion could also result in secretion of the PFP complex.
Considering the pivotal role of glucose and considering glycogen as another important source of glucose, 27,28 we also assayed the content of glycogen in isolated hepatocytes ( Figure S1D) iScience Article glycogen content of isolated toad hepatocytes gradually decreased during in vitro culture in both Glc + / Gln + /Pyr + and Glcˉ/Glnˉ/Pyrˉconditions, and decreased to a low level after 5 h' culture. After cultured in Glcˉ/Glnˉ/Pyrˉmedium for 5 h, hepatic glycogen decreased more as compared with that of Glc + /Gln + / Pyr + medium ( Figure S1D), and the expression of the PFP bg-CAT was upregulated ( Figure 1B). The degradation of glycogen during in vitro culture indicated that glycogen in the cells is not the main determinant of the nutritional state but rather the nutrients from outside the cell.
Taken together, these results revealed that the expression and secretion of bg-CAT are substantially upregulated under cell nutrient deficiency, and the PFP protein complex is an immediate responsive protein machine to cell nutrient deficiency. This phenomenon is well in accordance with our previous observation that the PFP complex in toad blood promptly responds to toad fasting. 24 AMPK signaling positively regulates the expression and secretion of bg-CAT under cell nutrient deficiency AMPK signaling is activated by a lack of energy or nutrients and switches on alternative catabolic pathways that generate ATP while switching off anabolic pathways and other processes that consume ATP. 29,30 Because toad cells secrete bg-CAT under nutrient deficiency (Figure 1), the expression and/or secretion of the PFP complex might be controlled by AMPK signaling. iScience Article Sequence alignment analysis of toad B. maxima AMPK a-subunits and acetyl coenzyme A carboxylase 1 (ACC1) on the basis of toad skin transcriptome 31 verified that the activation loop of AMPKs and phosphorylation sites (pAMPKa2 T172 and pACC1 S80 in human, respectively) were evolutionarily conserved from toad B. maxima to human ( Figure S2A). Activation of AMPK signaling in toad liver cells was observed under nutrient deficiency ( Figure S2B). Thus, we used two pharmacological AMPK signaling inhibitors, compound C and SBI-0206965 that act on the activation loop of AMPK a-subunit, 32,33 to examine the possible effects of AMPK signaling on bg-CAT regulation under cell nutrient deficiency. We first analyzed the cytotoxicity of these AMPK signaling inhibitors in toad liver cells. The results showed that dosages up to 10 mM compound C and 20 mM SBI-0206965 did not affect the viability of toad liver cells ( Figures S2C and S2D). The presence of compound C (2.5-5 mM) or SBI-0206965 (5-10 mM) did inhibit the activation of AMPK signaling in toad liver cells as observed by reduced phosphorylation of the canonical AMPK substrate ACC1 under Glcˉ/Glnˉ/Pyrˉconditions ( Figure S2E).
After validating the inhibitory effects of compound C and SBI-0206965 on AMPK signaling in toad liver cells, we next analyzed whether the expression and secretion of bg-CAT were regulated by AMPK signaling under cell nutrient deficiency. The mRNA levels of bg-CAT subunits BmALP1 and BmTFF3 were substantially decreased in the presence of compound C ( Figure 2A) or SBI-0206965 ( Figure 2E) under Glcˉ/Glnˉ/Pyrˉconditions. Moreover, the protein level of bg-CAT, as indicated by detecting its a-subunit BmALP1, was largely decreased in toad liver cells after treatment with 5 mM compound C ( Figure 2B) or 10 mM SBI-0206965 ( Figure 2F) for 3 h under Glcˉ/Glnˉ/Pyrˉconditions. Importantly, the treatment of toad liver cells with these two AMPK inhibitors substantially reduced bg-CAT secretion into the culture supernatant of the toad cells. Western blotting showed greatly reduced secretion of BmALP1 (a-subunit of bg-CAT) in culture supernatants of toad liver cells under Glcˉ/Glnˉ/Pyrˉconditions ( Figures 2C and 2G). The decreased hemolytic activity of the culture supernatants further confirmed that bg-CAT secretion was attenuated by these AMPK signaling inhibitors ( Figures 2D and 2H). Therefore, these results revealed that AMPK activation controls the expression and secretion of bg-CAT under glucose, glutamine, and pyruvate starvation as the main carbon source.
bg-CAT promotes extracellular protein import to facilitate intracellular amino acid supply and ATP production Because toad cells dispatch the PFP complex bg-CAT to the extracellular medium under nutrient deficiency downstream AMPK signaling (Figures 1 and 2), we next explored the possible cellular functions of bg-CAT under cell nutrient deficiency. It has been proposed that bg-CAT represents a novel PFP system-driven cell vesicular delivery, 13,23,24 which has been shown to stimulate cell pinocytosis/macropinocytosis to promote cellular material import including extracellular proteins dependent on the cell context and surroundings. 13,21,24 Therefore, we investigated the possible involvement of the PFP complex in mediating extracellular protein nutrient uptake under nutrient deficiency for cell energy supply and survival.
We firstly investigated whether bg-CAT bound extracellular substrates directly, the ovalbumin (OVA), toad B. maxima serum albumin (Bm-SA), and dextran (70 kDa), an indicator of macropinocytosis 34 were examined in a binding assay. The PFP bg-CAT complex showed a stronger interaction with extracellular proteins (OVA and Bm-SA) than with dextran in the BLI assay ( Figure 3A), and the K D values of bg-CAT with OVA and Bm-SA was approximately 2.33 3 10 À8 M and 2.47 3 10 À8 M, respectively. Ovalbumin-DQ (OVA-DQ) is a fluorescent indicator that fluoresces on proteolytic degradation. 35 Then, we used extracellular OVA-DQ to determine the possible bg-CAT-driven extracellular protein uptake and intracellular degradation. After treating toad liver cells with 100 nM bg-CAT, a striking increase in OVA-DQ fluorescence under Glcˉ/Glnˉ/Pyrˉconditions was observed by scanning confocal microscopy ( Figure 3B) and flow cytometry ( Figure 3C). bg-CAT showed no cytotoxicity in toad liver cells at dosages up to 400 nM under Glcˉ/Glnˉ/ Pyrˉconditions at 3 h ( Figure S3A), a level much higher than physiological concentrations (20-100 nM), 13 consistent with our previous study on peritoneal cells and urethral epithelial cells. This further emphasized bg-CAT as a physiological protein rather than merely a protein-peptide toxin. Furthermore, the augmented fluorescence of OVA-DQ under Glcˉ/Glnˉ/Pyrˉconditions was attenuated by immunodepletion of endogenous bg-CAT ( Figures 3B and 3C). Consistent with the results in toad liver cells, the addition of bg-CAT (40 nM) to mammalian HepG2 cells also promoted uptake and degradation of OVA-DQ under Glcˉ/Glnˉ/ Pyrˉconditions ( Figure S3B). Furthermore, colocalization of bg-CAT and OVA-DQ was readily observed, which exhibited a punctate pattern ( Figure 3D). Ethyl-isopropyl amiloride (EIPA), is a macropinocytosis inhibitor by inhibiting actin polymerization. 36  iScience Article internalization promoted by bg-CAT was inhibited in both toad liver cells ( Figure S3C) and mammalian HepG2 cells ( Figure S3D) in the presence of the inhibitor. These results suggested that bg-CAT might mediate extracellular protein intake in nutrient-deprived cells by inducing pinocytosis/macropinocytosislike endocytosis. Finally, bg-CAT also enhanced internalization of toad B. maxima serum albumin (Bm-SA) under Glcˉ/Glnˉ/Pyrˉconditions ( Figure S3E). Collectively, these results demonstrated that bg-CAT enhances uptake and intracellular degradation of extracellular proteins under cell nutrient deficiency.
Next, we further analyzed the metabolic situation of toad cells cultured in the presence of albumin under Glcˉ/Glnˉ/Pyrˉconditions for 7 h by LC/MS and LC-MS/MS. The levels of amino acids, including asparagine and glutamine, in toad liver cells treated with bovine serum albumin (BSA) and bg-CAT were augmented compared with those in cells treated with BSA only ( Figure S3F). Importantly, immunodepletion of endogenous bg-CAT reduced the levels of several amino acids in toad liver cells, including threonine, valine, and nonessential amino acids such as asparagine, aspartic acid, arginine, glutamine, glutamic acid, alanine, and serine ( Figure 3E). These results revealed that bg-CAT-mediated extracellular protein ingestion boosted the intracellular amino acid supply in toad cells under Glcˉ/Glnˉ/Pyrˉconditions.
To examine whether the increased amino acids were used for cell energy production, we analyzed total ATP availability in toad liver cells. 37 Luciferase-based ATP assessment showed that ATP concentrations were significantly decreased under Glcˉ/Glnˉ/Pyrˉconditions, but partially recovered after addition of extracellular proteins ( Figures 3F and S3G). Under Glcˉ/Glnˉ/Pyrˉconditions, the addition of purified bg-CAT to cultured toad cells augmented cellular ATP production in the presence of OVA ( Figure 3F) or BSA (Figure S3G). Moreover, immunodepletion of endogenous bg-CAT reduced ATP concentrations (Figures 3F iScience Article and S3G). Collectively, these results illustrated the capacity of bg-CAT to drive extracellular protein import under cell nutrient deficiency for cellular nutrient (amino acid) supply and ATP production.

bg-CAT supports toad cell survival in the presence of extracellular proteins under cell nutrient deficiency
Because bg-CAT was secreted by toad cells under nutrient deficiency (Figures 1 and 2), which mediates extracellular protein import and degradation, leading to increased amino acid supply and ATP production in nutrient-deprived toad cells (Figure 3), it was reasonable to postulate that this PFP complex bg-CAT could sustain the survival of nutrient-deprived cells in the presence of extracellular proteins.
The viability of toad cells was assessed by propidium iodide (PI) staining. 38 In toad liver cells, no difference in cell viability among the various culture conditions at the zero-time point (stained immediately after the cells mixed with different media) was observed and approximately 90% of the cells were viable as analyzed by PI staining (Figure S4A). However, toad liver cell death had increased substantially when the culture time was prolonged, and approximately 19% of liver cells had died after culture for 11 h under Glc + /Gln + /Pyr + conditions, while approximately 35% of liver cells had died under Glcˉ/Glnˉ/Pyrˉconditions ( Figure S4A). Notably, bg-CAT alone did not obviously affect the survival rate of toad liver cells under nutrient deficiency ( Figure S4A). However, the addition of bg-CAT did increase the survival rate of nutrient-deprived toad cells in the presence of extracellular proteins BSA ( Figure 4A) or ovalbumin (OVA) ( Figure 4B). Additionally, immunodepletion of endogenous bg-CAT greatly decreased the viability of nutrient-deprived toad cells (Figures 4C and 4D). It is of note that IgG slightly improved the cell survival, when comparing the control group between Figures 4A-4D, and this may be a result of potential interaction between bg-CAT and IgG, as IgG domain are often fused with pore-forming domain in other species. 10

bg-CAT is negatively regulated by high concentration of ATP in vitro
The above experimental evidence showed that toad cells secreted the PFP complex bg-CAT under nutrient deficiency to scavenge extracellular proteins for their nutrient supply, ATP production, and survival (Figures 1, 3, and 4). Accordingly, such a cellular macromolecular nutrient acquisition and intracellular digestion system should be tightly regulated. In addition to the positive regulation of bg-CAT expression and secretion by AMPK signaling (Figure 2), there should be specific negative feedback regulators of the bg-CAT-pathway. ATP, an end product of bg-CAT actions (Figure 3), might be a suitable candidate.
bg-CAT oligomerizes and forms channels (pores) on liposomes, which induces dye release from lipid vesicles, an advantageous model without ATP receptors. 12,39 Interestingly, the dye release due to bg-CAT channel formation on liposome was inhibited by 2.5 mM ATP or ADP, but not by AMP ( Figure 5A). The ATP concentration (2.5 mM) used was close to its physiological concentration in normal cells (5-10 mM). 40,41 The inhibition of dye release through bg-CAT channels by ATP (0.625-2.5 mM) was dependent . Continued (F) The ATP content in toad liver cells was determined by an ATP detection kit after incubation with 500 mg/mL OVA and 100 nM bg-CAT or 100 mg/mL anti-bg-CAT antibodies in Glcˉ/Glnˉ/Pyrˉmedium for 7 h. Rabbit IgG was used as an antibody control. Results (B, C, E, and F) are reported as the mean G SD of triplicate samples, ns (p R 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by the one-way ANOVA (B, C, and F) or unpaired t-test (E). All data are representative of at least two independent experiments. See also Figure S3. iScience Article on the concentration ( Figure 5B). Furthermore, a direct interaction between bg-CAT and ATP was observed by a surface plasmon resonance (SPR) assay with an apparent K D of approximately 2.8 3 10 À4 M under our assay conditions ( Figure 5C). Notably, oligomer formation of bg-CAT on liposome was not obviously changed in the presence of various ATP concentrations (0.625-2.5 mM) ( Figure 5D). This result suggested iScience Article that ATP at these concentrations did not obviously affect oligomerization or membrane insertion of bg-CAT. Finally, no ATPase activity was detected using purified bg-CAT even with a dosage up to 1 mM (Figure 5E). Collectively, these results revealed a direct interaction of ATP with bg-CAT and suggested that high concentrations of ATP (1-5 mM) may negatively regulate the opening state of bg-CAT channels.

DISCUSSION
PFPs are widely distributed in all kingdoms of life, which have long been recognized as either pore-forming toxin for microbial infection or host immune executors. 11,42,43 Specially, the knowledge of these PFPs derived from animals and plants is mainly focused on their roles in cell death. 44 The present study reported the direct and necessary function of toad B. maxima PFP protein complex bg-CAT in cell nutrient scavenging and energy supply under the deprivation of glucose and glutamine, essential nutrient components in cell metabolism. 2 We proposed an action model of bg-CAT in driving the uptake of extracellular macromolecules such as proteins for cell nutrient supply and survival under nutrient deficiency (graphical abstract). These findings provide further experimental evidence to support our previous hypothesis that toad B. maxima bg-CAT indeed acts as a novel system driven cell vesicular delivery that should play a physiological role in cell nutrient acquisition by mediating cellular nutrient import through endolysosomal pathways. 13,23,24 Although constitutively expressed in toad cells in nutrient-rich medium (Figure 1), bg-CAT was not secreted into the culture supernatant, indicating that the cells could acquire nutrients through membrane transporters under normal nutrient-rich environments. It is of note that BmALP1 protein level go down in normal media (Glc + /Gln + / Pyr + ) from 1 to 5 h ( Figure 1B), this may be a result of stable and/or higher nutrients level (especially glucose) in  (Figures 1A and 1B) was in accordance with a fact that cells decrease or even stop their protein production during nutrient deficiency. 45,46 However, the expression of bg-CAT was increased again afterward. Moreover, bg-CAT secreted into the culture supernatant was continuously augmented, corresponding to results that immune-depleted endogenous bg-CAT had more remarkable effect on amino acids supplement and cell survival under nutrient deficiency (Figures 3 and 4). And, the level of bg-CAT was readily attenuated by addition of glucose to the medium (Figure 1). Therefore, these results clearly illustrate that bg-CAT is a protein machine dispatched by toad cells in response to nutrient deficiency.
Plasma membrane sensors (extracellular sensors) and intracellular nutrient sensors, which survey the abundance of energy and major metabolites, play an important role in metabolic homeostasis and cell survival. 1,2,47 AMPK signaling is involved in sensing intracellular nutrient and energy availability, which is switched on by a lack of energy or nutrients. 48,49 Our assays employing pharmacological inhibitors suggested that AMPK signaling controls the expression and secretion of bg-CAT under cell nutrient deficiency ( Figure 2). These results are in accordance with the role of AMPK signaling in regulating cell responses to nutrient and energy deficiency. The present observations revealed that nutrient-deprived toad cells secrete a PFP machine such as bg-CAT downstream of AMPK signaling to scavenge and mediate cellular uptake of extracellular proteins for cell energy supply and survival. This PFP-driven vesicular delivery of extracellular nutrients represents an unknown strategy and mechanism involved in cell nutrient acquisition. Our data suggest that AMPK signaling is a positive regulator of the bg-CAT pathway, but the detailed molecular mechanisms are unclear at present stage, which is an intriguing question in future study.
Diverse endocytic pathways are available at the surface of metazoan cells. 3 Macropinocytosis is an actindependent endocytic pathway that non selectively engulfs materials, such as proteins and ions, mediated by epidermal growth factor, ion fluxes and ion channel, 7,50-52 bg-CAT can form channel in multiple cells and induces changes in ion fluxes. 12,14 In a murine dendritic cell (DC) model, bg-CAT enhances pinocytosis as determined by uptake of lucifer yellow, which enhances import of the antigen OVA. 21 bg-CAT also drives macropinocytosis in vivo and in vitro in toad osmoregulatory organs to facilitate toad water maintaining. 13 Consistently, pharmacological inhibitors in the present study also suggested that bg-CAT might promote uptake of extracellular proteins via classical pinocytosis/macropinocytosis-like endocytosis (Figures 3 and  S3). However, it is worth pointing out that the endocytic form mediating protein uptake in toad cells, including liver and gastrointestinal cells, stimulated by bg-CAT has not been completely clarified, especially in vivo. Indeed, the secreted bg-CAT could bind to ovalbumin and toad B. maxima serum albumin (nutrients) directly. Furthermore, the colocalization of bg-CAT and OVA-DQ was readily observed under cell nutrient deficiency. These results were suggested that the pathway of bg-CAT importing and enriching nutrients might be selective, which was different from classical pinocytosis/macropinocytosis-like endocytosis. 53 It is possible that bg-CAT may employ multiple cell entry mechanisms to scavenge protein nutrients depending on the cell context and surroundings, which is worthy of further investigation.
The functional diameter of bacterial toxin aerolysin channels is approximately 1.5 nm, which is large enough for translocation of oligonucleotides, peptides, and unfolded proteins. 54,55 As a member of af-PFPs, bg-CAT forms channels on membranes with a functional diameter similar to that of aerolysin. [12][13][14] Previously, it has been observed that the channels formed by bg-CAT served for the translocation of processed OVA peptides to cytosol for antigen presentation in murine DC cells. 21 Accordingly, the bg-CAT channels formed on endolysosomes of toad cells may act as channels to traverse amino acids and small peptides produced from imported proteins by proteolytic hydrolysis to cytosol for cell nutrient supply and energy (Figure 3).
The physiological concentrations of ATP in cells are estimated to be 5-10 mM. 40,56 Extracellular ATP levels are at the micromolar level (50-200 mM) under pathological conditions such as a tumor microenvironment, whereas in healthy tissues, extracellular ATP concentrations are sub-micromolar (likely about 10-100 nM). 41 bg-CAT did not possess an ATP-hydrolyzing activity ( Figure 5E). In liposome assays, the presence of ATP did not obviously affect oligomerization of bg-CAT, but dye release from bg-CAT channels formed in liposome was inhibited in a concentration-dependent manner by ATP (0.6-5 mM), but not by AMP. These results suggested that the normal intracellular contents of ATP could negatively regulate the opening state of bg-CAT channels ( Figure 5). In another word, bg-CAT may sense ATP abundance to regulate the opening state of its channels. bg-CAT, a PFP system is composed of membrane receptors, a negative regulator (BmALP3) and a positive regulator (FCGBP), which ll OPEN ACCESS iScience 26, 106598, May 19, 2023 iScience Article drives vesicles to respond to changes in nutrients. The pore-states of the PFP might be related to its system members. 16,17,23 Although liposome assay is well indicator for pore-states, 57 the complex regulation of pore-states induced by a PFP bg-CAT system is an intriguing question in future study by efficient and direct methods, such as lipid bilayer assay. 58,59 ATP is an energy situation marker in cells. The negative regulation of bg-CAT channels by high concentrations of ATP (1-5 mM) is in accordance with the fact that this PFP complex is secreted from nutrient-deprived cells to promote uptake of extracellular nutrients in response to a poor energy status (Figures 1  and 3). Furthermore, the concentration-dependent regulation of bg-CAT channels by ATP may lead to transcellular delivery of nutrients to internal tissue environments, such as tissue parenchymal cells by the release of nutrient exosomes. 24 bg-CAT is a complex of BmALP1 and BmTFF3, in which BmTFF3 acts as a chaperon and regulatory unit of BmALP1 to stabilize the PFP monomer and deliver it to proper targets. 16,17 bg-CAT undergoes oligomerization and forms channels on membranes. The inhibition of bg-CAT channels by ATP indicated that ATP binds to the PFP complex, which is in accordance with the binding of ATP to bg-CAT ( Figure 5C). However, at present, the exact binding sites of ATP on bg-CAT and the regulatory mechanisms are unknown and important future research directions. The findings that bg-CAT is positively regulated by AMPK signaling but negatively regulated by high concentration of ATP (1-5 mM) further emphasize and support the notion that the PFP complex is necessary for cell nutrient acquisition and metabolic flexibility.
It is well documented that autophagy is a cellular process to sequester and degrade intracellular components under nutrient deficiency, which is the last defense for cells under nutrient deficiency and excess autophagy may lead to cell death. [60][61][62] Comparatively, the SELC protein bg-CAT represents a novel cellular strategy to sense and uptake extracellular macromolecules like proteins as nutrients under cell nutrient deficiency. This novel cell nutrient acquisition pathway mediated by a secretory PFP such as bg-CAT through endolysosomal systems should be especially significant in the absence of essential small nutrient compounds, including glucose and glutamine, as revealed in the present study (graphical abstract). It may also be necessary and essential when classic plasma membrane-integrated transporters including solute carriers (SLCs) are absent such as in undifferentiated cells or they do not work properly. Cross-regulation of autophagy and pinocytosis/macropinocytosis is poorly understood. 8,63 The coordinative regulation between autophagy to sequence intracellular components and PFP-driven cell uptake to import extracellular compounds like that mediated by bg-CAT is worthy of further study.
SELC protein bg-CAT works in cell nutrient acquisition at least at two levels. First, toad B. maxima cells secrete bg-CAT to scavenge extracellular nutrients under nutrient deficiency at the cellular level (present study). Second, bg-CAT in toad B. maxima blood circulation is an immediate and active responsive element under toad fasting in vivo, which trans cellularly deliver and transport albumin-bound fatty acids to tissue parenchymal cells for their nutrient supply. 24 These findings uncovered the primary and necessary role of this PFP machine in toad B. maxima physiology for adaptation to various nutrient environments. Rationally, similar strategies and executive pathways should be conserved in vertebrates, in which various families of PFPs including af-PFPs are widely distributed. Knowledge from bg-CAT can provide clues to understand novel PFP-driven cell vesicular delivery systems in nutrient acquisition and metabolic flexibility. Although af-PFPs have not been clearly observed in Eutherian mammals, other PFP family members can readily compensate for the role of af-PFPs.
In conclusion, the present study elucidated that toad B. maxima cells secrete bg-CAT, a PFP and trefoil factor (TFF) complex assembled depending on environmental cues under glucose, glutamine, and pyruvate deficiency. This PFP complex supports cell survival by driving the cellular import of extracellular proteins through endolysosomal pathways. The imported proteins serve as nutrients in nutrient-deprived cells for energy supply. AMPK signaling positively regulates the expression and secretion of bg-CAT, whereas high concentrations of ATP (> 1 mM) bind to and negatively regulate bg-CAT channels. Our findings define the essential role of toad B. maxima PFP complex bg-CAT in cell macromolecular nutrient scavenging, providing a new paradigm for PFPs in cell nutrient acquisition and metabolic flexibility.

Limitations of the study
Our work was restricted in vitro cell experiments. Consequently, it is necessary to study whether the secretory PFP bg-CAT contribute to the nutrient acquisition in vivo under starvation, which revealed that the PFP bg-CAT was secreted into toad blood in response to toad fasting, 24  iScience Article supported cell survival. Further research should also elucidate the mechanism underlying the AMPK signaling regulated the expression and secretion of PFP bg-CAT under nutrient deficiency. Moreover, the PFP system, consisted with receptor and regulator, acts along cell endocytic and exocytic pathways with channel formation on endolysosomes and performs multiple functions including immune and tissue repair, 16,17,23,24 its channel characteristic and regulation of channel-states are worth for further study.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yun Zhang (zhangy@mail.kiz.ac.cn).

Material availability
All unique/stable reagents generated in this study are available from the lead contact with bg-CAT protien and anti-bg-CAT antibodies.
Data and code availability d Original western blot images reported in this paper will be shared by the lead contact upon request.
d Our study not generated any original code.
d Any additional information required to reanalyze the data reported in this paper is available form the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Animal
Feeding of toads (B. maxima) was performed as described previously and the males with a mean body weight of 25 G 5 g were used for this study (Li et al., 2017). All procedures and the care and handing of animals were approved by the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (Approval ID: IACUC-OE-2021-05-001).

Cell culture
Toad liver cells were isolated by a two-step EDTA/collagenase perfusion technique as previously described with the following modifications. 64 The perfusion solution Ringer's buffer and perfusion solution II containing 1 mg/mL collagenase (Solarbio, Cat C8140) were used for toad liver tissue perfusion. After perfusion, liver tissues from three toads were cut into pieces and washed with Ringer's buffer once and then oscillatory digested in perfusion solution II at 26 C for 1 h. The cells were filtered through a 40-mm mesh and collected by centrifugation at 805 g for 5 min at 4 C. Toad stomach and intestinal cells were isolated as described previously. 13 The mammalian cell line HepG2 was purchased from Kunming Cell Bank, Chinese Academy of Sciences. Cells were cultured in DMEM/F-12 (Biological Industries, Cat 01-172-1A) containing 10% fetal bovine serum (Biological Industries, Cat 04-001-1A) and 1% Penicillin-Streptomycin Solution (Biological Industries, Cat 03-031-1B) at 37 C with 5% CO 2 .