Autophagy, the evolutionarily conserved pathway to target unwanted or damaged cellular contents to lysosomes for degradation, is linked to numerous human diseases (Levine and Kroemer, 2019; Mizushima and Levine, 2020) and considered a therapeutic target (Galluzzi et al., 2017).

Autophagy can be activated by many approaches, ranging from amino acid starvation, caloric restriction, physical exercises, to treatment with chemicals by known or unknown mechanisms (Galluzzi et al., 2017). Some chemicals that induce autophagy are under clinical trials for neurodegenerative diseases, cancers, autoimmune disorders or metabolic diseases, including (but not limited to) rapamycin, idalopirdine, SB-742457, metformin, resveratrol, lithium, spermidine, and trehalose (Galluzzi et al., 2017; Mizushima and Levine, 2020; Suresh et al., 2020). Among these autophagy activators, rapamycin is widely used in basic and translational research; it inactivates mechanistic target of rapamycin complex 1 (mTORC1), the nutrient-sensing kinase complex that promotes anabolism and inhibits catabolism, mainly autophagy (Saxton and Sabatini, 2017). However, specific autophagy activators that have no or limited impact on other pathways are lacking for research needs and for potential clinal use.

In 2013, Beth Levine lab reported an autophagy-inducing Tat-BECN1 peptide (hereinafter referred as to Tat-BECN1), derived from Beclin 1 region that is required for HIV-1 Nef binding (Shoji-Kawata et al., 2013). Beclin 1 is a subunit of both Class III PI3K complexes, where Class III PI3K-C1 is regarded as autophagy initiation complex for autophagosome nucleation, and Class III PI3K-C2 mediates autophagosome maturation, endocytosis and LC3-associated phagocytosis (LAP) (Levine et al., 2015; Hurley and Young, 2017). Upon Tat-BECN1 treatment, autophagy induction was observed in 10 cell lines and primary cultures (Table 1); when Tat-BECN1 was administrated to mice intraperitoneally, autophagy induction was observed in heart, skeletal muscle (vastus lateralis) and pancreas (Shoji-Kawata et al., 2013). Tat-BECN1 showed protective effects in several in vitro and in vivo infection models of Sindbis virus, chikungunya virus, West Nile virus (WNV), HIV-1, L. monocytogenes (Shoji-Kawata et al., 2013). It also enhanced the degradation of small mutant huntintin protein aggregates in cells, indicating a therapeutic potential for neurodegenerative diseases (Shoji-Kawata et al., 2013).

In this review, we analyze the applications of Tat-BECN1 in around 100 studies since its discovery, discuss the current understanding of how it functions, the strategies to improve its activity, and the impact of Tat-BECN1 on pathophysiology. The modifications of Tat-BECN1 are discussed in details in OPTIMIZATION AND DEVELOPMENT session; in other sessions, Tat-BECN1 is used to refer to the agent, regardless of being the original or modified version.

Tat-BECN1 and modified versions have been employed by around 100 studies and shown robust induction of autophagy in a variety of cellular and organismal models. Tat-BECN1 can be directly applied to cultured cells, isolated tissues and embryos, injected intraperitoneally, intravenously, hippocampally, or infused directly to rodent animals. It is soluble in aqueous solution. Depending on cell or tissue type, the concentration and duration of treatment may vary. Other than the cells reported in the original study (Shoji-Kawata et al., 2013), around 40 cell lines or primary cell cultures respond to Tat-BECN1 for autophagy activation by experimental validation (Table 1). In addition to cardiac muscle, skeletal muscle and pancreas tissues reported in the original study (Shoji-Kawata et al., 2013), rodent heart (Shirakabe et al., 2016; An et al., 2017; Sun et al., 2018; Tong et al., 2019; Nah et al., 2020), brain (intraperitoneal injection (Li et al., 2016; He et al., 2017; Zhang et al., 2017; Forte et al., 2020; Kim et al., 2021), hippocampal injection (Glatigny et al., 2019; De Risi et al., 2020), intravenous injection (Luo et al., 2018), direct infusion (Shehata et al., 2018), intracerebroventricular injection (He et al., 2019)), spinal cord (He et al., 2016), eye (eye drop) (Kasetti et al., 2021), kidney (Livingston et al., 2019; Shi et al., 2020), liver (Soria et al., 2018; Soria et al., 2021), lung (Nikouee et al., 2021), bone (Cinque et al., 2015; Bartolomeo et al., 2017), articular cartilage (intra-articular injection) (Wang F. et al., 2019; Rockel et al., 2020), ovary (Watanabe et al., 2020), tumor xenografts of breast cancer cells (Wang et al., 2015a; Wang et al., 2015b; Vega-Rubín-De-Celis et al., 2018; Zhou et al., 2019), fibrosarcoma cells (Pietrocola et al., 2016) or ovarian cancer cells (Ding et al., 2018), as well as zebrafish embryos (Zhu et al., 2017), show increased autophagy level upon Tat-BECN1 treatment (Table 1; Figure 1A). Table 1 also summarizes cells and tissues that show responses to Tat-BECN1 while autophagy induction is not tested.

To understand the molecular mechanisms by which Tat-BECN1 exerts its effects will not only help elucidate the functionality of autophagy activation in physiology and pathology, but also shed light on how autophagy activators can be further optimized and designed. Here we discuss the progress of mechanistic studies of Tat-BECN1 (Figure 1B).

On-going optimization and development of Tat-BECN1 aims to improve activity, solubility, membrane permeability and stability, as well as targeted delivery (Figure 1C).

As discussed above, Tat-BECN1 is widely tested as an autophagy activator for basic and translational research (Table 1), and shows great therapeutic potential in cancers, neurodegenerative diseases, infectious diseases, injury recoveries, aging and so on. Here we try to compare the effects of Tat-BECN1 that impact pathophysiology from three aspects, through mediating autophagic degradation or autophagy-dependent secretion, through promoting autophagic cell death or preventing apoptotic cell death, and through alleviating stress or augmenting stress (Figure 1D), in order to offer a comprehensive view of Tat-BECN1 application across different pathophysiological models.

Tat-BECN1 can exert effects by inducing autophagic degradation, including general autophagy and selective autophagy (i.e., xenophagy, virophagy, mitophagy, lipophagy, ER-phagy and aggrephagy). Tat-BECN1 enhances bacterial clearance, during uropathogenic E. coli infection in mouse bladder (Miao et al., 2015), M. tuberculosis infection in mouse bone marrow-derived macrophages (BMDMs) (Franco et al., 2017), K. pneumoniae infection in mouse lungs (Nikouee et al., 2021) and opportunistic infection of M. tuberculosis or M. avium in HIV-1-infected human macrophages (Sharma et al., 2021). Tat-BECN1 protects against viral infections, by inhibiting HIV replication in human monocyte-derived macrophages (MDMs) (Zhang et al., 2018) and memory CD4⁺ T cells (Zhang et al., 2019), and by inhibiting Middle East respiratory syndrome coronavirus (MERS-CoV) replication in VeroB4 cells (Gassen et al., 2019). Tat-BECN1 activates mitophagy in rodent heart and protects against pressure-overload-induced heart failure (Shirakabe et al., 2016) and high fat diet-induced diabetic cardiomyopathy (Tong et al., 2019), in kidney and protects against renal ischemia/reperfusion injury (Livingston et al., 2016; Livingston et al., 2019), in brain and kidney and prevents high salt diet-induced hypertension-related stroke occurrence (Forte et al., 2020); it prevents mitochondrial dysfunction in skeletal muscles and the appearance of markers of muscle fiber denervation caused by prolonged mTORC1 loss (Baraldo et al., 2020). Tat-BECN1 induces lipophagy of acetylated low density lipoprotein-induced lipid droplets in macrophages; interestingly, it increases the biogenesis of oleic acid-induced lipid droplets (Hadadi-Bechor et al., 2019). Tat-BECN1 triggers degradation of amyloid fibrils in Alzheimer’s disease (Luo et al., 2018) and middle-aged (De Risi et al., 2020) brains and rescues memory deficits, removes ubiquitinated protein aggregates induced by WNV infection (Kobayashi et al., 2020), relieves abnormal cytosolic and nuclear glycogen storage in liver with distal urea cycle disorders (Soria et al., 2021), clears glaucoma-causing mutant myocilin and reduces elevated intraocular pressure (Kasetti et al., 2021), enhances FAM134B-mediated ER-phagy of misfolded procollagen in chondrocytes (Forrester et al., 2019). Other than protein quality control, Tat-BECN1 promotes degradation of synaptic proteins and erasure of reconsolidation-resistant fear memories (Shehata et al., 2018), stabilizes microtubules in neurons by targeting microtubule-destabilizing protein and accelerates axon regeneration after spinal cord injury (He et al., 2016), lowers cardiac lipoprotein lipase and triglyceride accumulation and restores cardiac function in obese mice (An et al., 2017), increases ureagenesis in liver by providing key urea-cycle intermediates and improves ammonia clearance in hyperammonemia model (Soria et al., 2018), reverts the accumulation of oxidized proteins like CaMKII and sinus node dysfunction induced by insulin sensitizers (Woo and Kim, 2021), downregulates β-catenin pathway and regulates chondrogenic fate specification of cranial neural crest cells (Yang et al., 2021) while downregulates Notch1 which counteracts β-catenin in bone marrow stromal cells (Choi et al., 2018), decreases the level of pluripotency factors POU5F1, NANOG and SOX2 in cancer stem cells (Sharif et al., 2017), decreases fibrotic markers and inhibits lung fibroblast to myofibroblast differentiation (Sosulski et al., 2015).

Emerging evidence indicates that autophagy pathway regulates secretion of specific cargos instead of lysosomal degradation (New and Thomas, 2019). Tat-BECN1 increases the release of insulin in murine and human primary islets (Goginashvili et al., 2015) (Tat-BECN1 is also reported to reduce adenosine-stimulated insulin secretion in islets (Israeli et al., 2018)), the secretion of candidate protein biomarkers of tumor cell autophagy (Kraya et al., 2015), and the production of monoclonal antibody in CHO cells (Braasch et al., 2021). Tat-BECN1 restores collagen secretion from chondrocytes to extracellular matrix and rescues bone growth in mice with lysosomal storage disorders (Bartolomeo et al., 2017) or mice with FGF signaling defects (Cinque et al., 2015). Tat-BECN1 attenuates extracellular matrix degradation in osteoarthritis chondrocytes (Wang F. et al., 2019; Wu et al., 2020) and ameliorates cartilage degeneration in rodent osteoarthritis model (Wang F. et al., 2019). Future work is needed to elucidate what mechanisms determine the destination of such cargos.

Prolonged Tat-BECN1 treatment leads to autosis, an autophagy-dependent, apoptosis and necrosis-independent cell death, mediated by Na⁺/K⁺ ATPase pump (Liu et al., 2013; Fernández et al., 2020). Tat-BECN1 induces autosis in HIV-infected human MDMs (Zhang et al., 2018) and memory CD4⁺ T cells (Zhang et al., 2019), but not in uninfected counterparts; it sensitizes primary renal tubular cells to TGFβ1-induced non-apoptotic cell death (Livingston et al., 2016). Tat-BECN1 has been assessed for systemic toxicity in vivo, which is negligible (Shoji-Kawata et al., 2013; Wang et al., 2015a; Ding et al., 2018). However, it exhibits cytotoxicity in cancer cells (Wang et al., 2015a; Ding et al., 2018; Song et al., 2018; Zhou et al., 2019) and cancer stem-like cells (Sharif et al., 2017; Sharif et al., 2019), reduces the growth of tumor xenografts in vivo (Wang et al., 2015a; Ding et al., 2018; Song et al., 2018; Vega-Rubín-De-Celis et al., 2018; Zhou et al., 2019). Tat-BECN1 worsens the brain damage after cerebral ischemic stroke (He et al., 2017; Zhang et al., 2017; He et al., 2019) and abolishes the neuroprotective effects of such agents as ganglioside GM1 (Li et al., 2016), puerarin (He et al., 2017) and breviscapine (Zhang et al., 2017), likely due to aggravating inflammatory responses (He et al., 2019). What confer the sensitivity of HIV-infected cells, cancer cells and ischemic neurons to Tat-BECN1-induced cytotoxicity? How does Tat-BECN1 affect tumor growth, by autosis, ferroptosis (Song et al., 2018) or T cell-mediated immune response (Pietrocola et al., 2016)? Can Tat-BECN1 be modified for targeted delivery? Future research will be required to address these questions.

When autosis is already induced in cardiomyocytes during ischemia/reperfusion, Tat-BECN1 exacerbates myocardial injury (Nah et al., 2020); while administration of Tat-BECN1 prior to ischemia/reperfusion (Nah et al., 2020) or at the time of reperfusion (Xie et al., 2021) reduces cell death and protects cardiac function. This suggests that the timing of intervention is important; autophagy activation at early stage of a developing condition may be beneficial. Tat-BECN1 inhibits apoptotic cell death in many in vitro or in vivo models: during renal ischemia/reperfusion, pretreatment with Tat-BECN1 inhibits apoptosis and prevents renal injury (Livingston et al., 2019); under oxygen glucose deprivation, the in vitro model of ischemia, Tat-BECN1 decreases apoptosis of endothelial progenitor cells (Jiang et al., 2020) and retinal neurons (Mathew et al., 2021); Tat-BECN1 blocks cardiomyocytes apoptosis under pressure overload (Shirakabe et al., 2016); it also alleviates cisplatin (a cancer drug known for nephrotoxicity)-induced cell death in renal proximal tubule cells (Wang S. et al., 2021) or PKM2 silence-induced apoptosis in acute myeloid leukemia cell line with mutated nucleophosmin (Wang L. et al., 2019). Tat-BECN1 not only reduces apoptosis but enhances proliferation in synovial intima cells leading to synovial hyperplasia in mouse knee joints (Rockel et al., 2020); it augments cardiac hypertrophy in autosomal dominant polycystic kidney disease mouse model (Atwood et al., 2020). Tat-BECN1 attenuates apoptosis and necrosis, in brain endothelial cells and renal epithelial cells exposed to high salt (Forte et al., 2021), in human neuroblastoma cells infected with WNV (Kobayashi et al., 2020). Tat-BECN1 preserves brain structures and sensorimotor functions after neonatal hypoxic-ischemic injury (Kim et al., 2021), protects renal proximal tubule cells against phosphotoxicity induced by high phosphate and increases urinary phosphate excretion in mice (Shi et al., 2020), rescues Aldehyde dehydrogenase 5a1-deficient mice from premature lethality (Vogel et al., 2016), increases survival rate of zebrafish embryos with anthracycline-induced cardiotoxicity (Wang Y. et al., 2021). Administration of Tat-BECN1 at neonatal stage upregulates the number of primordial follicles even at middle-age and extends the fertility and reproductive lifespan of female mice (Watanabe et al., 2020).

Autophagy is a major pathway to respond to stress and maintain homeostasis. In most examples discussed in the sessions above, Tat-BECN1 exhibits beneficial effects to cells or organisms under stress conditions, like protections against bacterial and viral infections, recoveries from injuries, detoxication of aberrant metabolism. A few exceptions include that Tat-BECN1 increases unexpected synovial hyperplasia (Rockel et al., 2020) and cardiac hypertrophy (Atwood et al., 2020); it intensifies brain damage after cerebral ischemic stroke (Li et al., 2016; He et al., 2017; Zhang et al., 2017; He et al., 2019). On the contrary, in neonatal hypoxia-ischemia model (Kim et al., 2021) or high salt diet-induced hypertension-related stroke model (Forte et al., 2020), Tat-BECN1 prevents brain damage; it also improves the formation of long-term spatial memory (Hylin et al., 2018) or novel memory (Glatigny et al., 2019) in young rodents, rescues age-related memory decline in middle-aged mice (Glatigny et al., 2019; De Risi et al., 2020). How brain tissues respond to Tat-BECN1 treatment in different models shall be further investigated.

In addition to the abovementioned scenarios, during endotoxemia induced by lipopolysaccharide (LPS) or sepsis induced by pneumonia, Tat-BECN1 alleviates inflammation and reduces injuries in heart (Sun et al., 2018; Sun et al., 2021) and lung (Nikouee et al., 2021); it suppresses neutrophil infiltration into acutely inflamed mouse tissues (Reglero-Real et al., 2021). In undernourished aged mice, Tat-BECN1 decreases the plasma levels of the glucogenic amino acid and restores the blood glucose levels to maintain energy homeostasis (Nahata et al., 2021), in line with the beneficial effects of Tat-BECN1 in age-related decline in memory (Glatigny et al., 2019; De Risi et al., 2020) or fertility (Watanabe et al., 2020). Tat-BECN1 mediates cell differentiation and fate determination (Sosulski et al., 2015; Sharif et al., 2017; Choi et al., 2018; Yang et al., 2021). It is reported to affect the length of primary cilia; however, the effect is controversial (Wang S. et al., 2015; Iaconis et al., 2020; Wang S. et al., 2021).

As discussed above, Tat-BECN1 is a potent autophagy activator and a powerful tool to investigate the role of autophagy in various cells and tissues; optimal treatment conditions, especially timing, concentration/dosage and duration, shall be tested in cells or tissues of interest beforehand. Tat-BECN1 is likely to function independent of mTORC1 and a side-by-side comparison of Tat-BECN1 and mTORC1 inhibitors is recommended.

A thorough understanding of the molecular mechanisms underlying Tat-BECN1-induced autophagy is of importance. So far, GLIPR2 (Shoji-Kawata et al., 2013; Zhao et al., 2021) and Beclin 1 in Class III PI3K complexes (Chang et al., 2019b; Zhao et al., 2021) are experimentally confirmed Tat-BECN1 targets. GLIPR2 is a negative regulator of autophagy interacting with Tat-BECN1 and Beclin 1 via BARA domain β-sheet 1 (Shoji-Kawata et al., 2013; Li et al., 2017; Zhao et al., 2021); it will be interesting to test whether Rubicon, a well-known negative regulator of autophagy, which also interacts with Beclin 1 via BARA domain β-sheet 1 (Chang et al., 2019b), is another target of Tat-BECN1. Class III PI3K-C1 (autophagy initiation) and -C2 (autophagosome maturation, endocytosis, LAP) complexes (Levine et al., 2015; Hurley and Young, 2017) are not discerned by Tat-BECN1 for activation, at least in vitro (Chang et al., 2019b), which may explain why Tat-BECN1 triggers autophagy-independent effect such as endocytosis (Wong et al., 2019). Further investigation on Class III PI3K complexes and regulators may facilitate the design of Class III PI3K-C1 or -C2-specific Tat-BECN1.

Advancement in chemical biology and materials science will accelerate the optimization and development of Tat-BECN1 with better bioactivity and efficacy. Additional work is required to determine long-term and systemic effects of Tat-BECN1 treatment in vivo, before moving forward to test Tat-BECN1 as novel clinical therapeutics.