Classical PROTACs rely on E3 ubiquitin ligases and the proteasome to eliminate intracellular target proteins, but they are difficult to apply to extracellular proteins, large-scale cellular components, and bacterial proteins. LYTAC, ATTEC, AUTAC, and BacPROTAC respectively utilize endocytosis–lysosome, autophagy–lysosome, or prokaryotic protease systems to establish targeted clearance routes for targets with different spatial locations and physical scales.

Keywords: PROTAC; targeted protein degradation; LYTAC; ATTEC; AUTAC; BacPROTAC; ClpCP; autophagy–lysosome

1 Degradation Basis and Functional Boundaries of Classical PROTACs

1.1 Substrate-Processing Logic of the Ubiquitin–Proteasome System

(1) Ubiquitin Tagging and Proteasomal Recognition

The ubiquitin–proteasome system (UPS) sequentially carries out ubiquitin activation, transfer, and substrate selection through E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. After a target protein acquires a polyubiquitin signal suitable for recognition by the 26S proteasome, it undergoes deubiquitination, ATP-dependent unfolding, and polypeptide-chain translocation before entering the proteolytic core for hydrolysis. E3 ligases perform the critical substrate-selection function and therefore represent the key molecular node that allows PROTACs to redirect the UPS.

(2) Reprogramming E3 Substrate Selection by PROTACs

PROTACs use bifunctional structures to induce a new spatial proximity between a target protein and a specific E3 ligase, thereby providing ubiquitination opportunities for proteins that are not naturally recognized as substrates of that E3. Their pharmacological effects do not depend on continuous occupancy of the target protein’s functional site but on successfully triggering a “target recruitment–ubiquitination–proteasomal clearance” event. Therefore, evaluation of PROTAC activity requires not only ligand-binding analysis but also confirmation of target-protein depletion and its dependence on the corresponding E3 ligase and proteasome.

1.2 POI–PROTAC–E3 Ternary Complex

(1) Three Structural Modules

Classical PROTACs generally consist of a target-protein ligand (POI ligand), a Linker, and an E3 ligase ligand. The POI ligand determines the target-recognition range, the E3 ligand determines which ubiquitin ligase is recruited, and the Linker regulates the distance, relative orientation, and conformational freedom between the two proteins. The Linker is not merely a connecting unit; its length, rigidity/flexibility, chemical composition, and attachment site can all substantially alter the conformation of the ternary complex.

(2) Affinity and Degradation Efficiency

High binary affinity does not necessarily correspond to high degradation efficiency. After formation of the ternary complex, the spatial accessibility of ubiquitinatable lysines on the target surface, the relative position of the E2–E3 catalytic region and the target protein, complex lifetime, and efficiency of ubiquitin-chain extension can all affect final degradation. Thus, the core of PROTAC optimization is not only to improve ligand binding but also to obtain a ternary-complex geometry favorable for ubiquitin transfer and subsequent proteasomal processing.

1.3 Cooperativity and the Hook Effect

(1) Ternary-Complex Cooperativity

When the POI and E3 ligase enter the same ternary complex, additional protein–protein interactions may form between their surfaces. Their influence is commonly described by the cooperativity parameter α. α>1 indicates positive cooperativity, meaning that the ternary complex is more stable than predicted from independent binary interactions; α<1 indicates negative cooperativity. Linker length, attachment site, exit vector, and complementarity of protein interfaces can all alter cooperativity and are important parameters in PROTAC structural optimization.

(2) Hook Effect

At excessively high PROTAC concentrations, the POI and E3 may be occupied separately by different PROTAC molecules, increasing the proportions of POI–PROTAC and PROTAC–E3 binary complexes while reducing the proportion of productive POI–PROTAC–E3 ternary complexes. This can produce a bell-shaped concentration–degradation relationship in which degradation first increases and then decreases, known as the hook effect. Dose evaluation should therefore cover low, medium, and high concentration ranges to avoid underestimating true degradation activity based only on high-concentration results.

2 Need for Targeted Degradation Beyond the Proteasome

2.1 Substrate-Scale Limitations of the Proteasome

(1) Processing of Individual Protein Substrates

The proteolytic core of the 26S proteasome has a restricted entry channel. Target proteins usually need to be recognized and undergo ATP-dependent unfolding before entering the catalytic chamber as unfolded polypeptide chains. Therefore, the proteasome is more suitable for substrates that can be individually recognized, mechanically unfolded, and continuously translocated.

(2) Clearance of Large-Scale Cellular Components

Protein aggregates, lipid droplets, membranous structures, and intact organelles cannot enter the proteasomal core as whole structures. Autophagosomes can accommodate larger substrates through membrane expansion and sequestration and then complete degradation after fusion with lysosomes. Therefore, large protein complexes, aggregates, and organelles are more suitable for autophagy–lysosome-mediated clearance.

2.2 Spatial Limitations of Extracellular Targets

E3 ligases and the 26S proteasome are mainly distributed in the cytoplasm and nucleus, whereas secreted proteins and extracellular domains of membrane proteins are located outside the cell. For membrane proteins such as EGFR and PD-L1, even high-affinity extracellular ligands cannot directly establish a classical POI–PROTAC–E3 complex. These targets need to enter the endosomal system through receptor-mediated endocytosis and subsequently be delivered to lysosomes. Therefore, the core design variable shifts from E3 selection to endocytic-receptor selection and regulation of intracellular trafficking.

2.3 Absence of the Classical Eukaryotic UPS in Prokaryotic Cells

(1) Limitations of the Traditional PROTAC Pathway

Most bacteria do not possess a complete eukaryotic E3–ubiquitin–26S proteasome system. Therefore, the E3 recruitment, ubiquitination, and proteasomal recognition pathway required by traditional PROTACs cannot be directly applied to prokaryotic cells.

(2) Alternative Role of Prokaryotic Protease Systems

Bacteria possess ATP-dependent protein quality-control systems such as Clp and Lon. Clp ATPases are responsible for substrate recognition, unfolding, and translocation, while proteolytic cores perform subsequent hydrolysis. BacPROTAC uses these endogenous prokaryotic proteases to extend the principles of induced proximity and substrate redirection from E3 ligases to Clp protease systems.

3 LYTAC-Mediated Degradation of Extracellular and Membrane Proteins

3.1 Endocytic Receptor-Mediated Target Delivery

(1) Bridging of Target and Endocytic Receptor

One end of a LYTAC (lysosome-targeting chimera) recognizes an extracellular protein or cell-surface target, while the other end binds a cell-surface receptor capable of internalization. After formation of a target–LYTAC–receptor complex, the entire complex enters receptor-mediated endocytosis, allowing the extracellular target to enter the intracellular vesicular trafficking system.

(2) Endosome–Lysosome Trafficking

After the complex enters early endosomes, it is exposed to progressively acidified environments as the endosome matures. Some trafficking receptors can recycle back to the cell surface, whereas the target protein continues toward late endosomes and lysosomes, where it is ultimately degraded by cathepsins and other acidic hydrolases. LYTAC therefore exploits the endogenous “surface receptor–endosome–lysosome” transport axis rather than the cytosolic UPS.

3.2 Receptor Types and LYTAC Applicability

(1) CI-M6PR System

CI-M6PR possesses receptor-mediated internalization and lysosomal-trafficking functions and can therefore serve as a relatively broadly applicable LYTAC-recruiting receptor. Degradation efficiency depends not only on target and receptor affinity but also on receptor-binding valency, endocytic efficiency, intracellular sorting, and receptor recycling capacity.

(2) ASGPR System

ASGPR is highly expressed in hepatocytes and can support efficient internalization through glycan ligands, making it suitable for construction of hepatocyte-biased LYTACs. Receptor selection therefore determines not only whether the target can enter lysosomes but also the potential cellular and tissue selectivity of the degrader.

3.3 Determination of LYTAC-Mediated Degradation

A reduction in cell-surface target signal alone cannot prove target-protein degradation because receptor-mediated internalization itself can reduce surface signal. A more complete mechanistic evidence chain should include surface-target internalization, reduction in total cellular target protein, and rescue of degradation after lysosomal-function blockade. If the surface level decreases while total protein remains largely unchanged, intracellular trafficking or redistribution is more strongly supported than terminal degradation.

4 ATTEC- and AUTAC-Mediated Autophagy-Targeted Clearance

4.1 ATTEC Directly Recruits Targets to LC3

(1) LC3 as an Autophagosome Recruitment Node

After lipidation, LC3-family proteins form membrane-associated LC3-II and localize to autophagosomal membranes, serving as an important node in autophagosome formation and cargo recognition. ATTEC (autophagosome-tethering compound) simultaneously binds a target and LC3, increasing spatial proximity between the target and the autophagic membrane and facilitating target entry into the autophagosome followed by lysosomal delivery after maturation.

(2) Clearance of Abnormal Proteins

Mutant huntingtin (mHTT) is a representative ATTEC research target. ATTEC binds mHTT through its target-recognition moiety and LC3 through another functional moiety, thereby increasing the probability that the abnormal protein enters autophagosomes. If the target ligand can distinguish mutant from wild-type HTT, selective clearance may be further achieved.

(3) Clearance of Non-Protein Structures

ATTEC can also be applied to non-protein cellular structures such as lipid droplets. A lipid-droplet-targeting molecule can bind lipid droplets at one end and recruit LC3 at the other, thereby promoting entry of lipid droplets into lipophagy. These studies demonstrate that ATTEC targets are not limited to individual proteins and can extend to cellular structures with recognizable interfaces.

4.2 AUTAC Alters Target Fate Through Selective Autophagy

(1) Selective Autophagy Tag

The major difference between AUTAC (autophagy-targeting chimera) and ATTEC is that AUTAC does not primarily rely on direct target–LC3 bridging. Representative AUTACs use autophagy-inducing tags to alter the quality-control state of the target, making it more readily recognized by the selective autophagy machinery and subsequently delivered into autophagosomes.

(2) K63-Linked Ubiquitin Signals

Some AUTAC systems can promote formation of K63-linked ubiquitin signals near the target. These ubiquitin chains differ functionally from ubiquitin signals mainly used for proteasomal delivery in classical PROTACs and are more involved in selective autophagy recognition, potentially connecting to LC3-positive autophagic membranes through autophagy receptors such as SQSTM1/p62. Thus, non-proteasomal degradation does not necessarily mean that ubiquitin is completely absent from the process; rather, the terminal degradation system is no longer the 26S proteasome.

(3) Organelle Clearance

AUTAC can further be used for large organelles such as damaged mitochondria. Intact mitochondria cannot enter the 26S proteasome but can be engulfed by autophagic membranes and cleared through mitophagy after lysosomal delivery. Evaluation of organelle degradation should focus on changes in total organelle mass or number rather than relying solely on changes in a single mitochondrial protein or membrane potential.

4.3 Mechanistic Differences Between ATTEC and AUTAC

5 BacPROTAC-Mediated Reprogramming of Bacterial Clp Proteases

5.1 Substrate-Processing Mechanism of Clp Proteases

(1) Recognition and Unfolding Functions of ClpC/ClpC1

Clp-family proteases generally consist of an AAA+ ATPase and a proteolytic core. ClpC or ClpC1 recognizes and binds substrates and uses ATP hydrolysis to generate mechanical force, gradually unfolding folded proteins into polypeptide chains that can enter the proteolytic core.

(2) Proteolytic Functions of ClpP/P1P2

ClpP and the mycobacterial P1P2 protease core contain restricted hydrolytic chambers. Intact folded proteins generally cannot enter directly and require ClpC/ClpC1-mediated capture, unfolding, and translocation. Therefore, BacPROTAC mainly reprograms the substrate-recognition entry point of the Clp system rather than directly altering the catalytic center of ClpP.

5.2 pArg-Mediated ClpCP Substrate Recognition

(1) pArg Quality-Control Tag

In some Gram-positive bacteria, phosphoarginine (pArg) can function as a protein quality-control tag recognized by the N-terminal domain of ClpC and directed to ClpCP. Its similarity to eukaryotic ubiquitin lies in its ability to provide substrate-selection information to a degradation system, although its molecular structure and recognition mechanism are entirely different.

(2) mSA Proof of Concept

Early BacPROTAC studies linked biotin, which binds monomeric streptavidin (mSA), to a ClpC-recognition moiety, thereby artificially recruiting mSA to ClpCP for proteolysis. This system demonstrated that bifunctional molecules can alter the substrate-recognition range of prokaryotic proteases and confer degradability on proteins that are not natural ClpCP substrates.

5.3 BacPROTAC Ternary Complex

(1) POI-Recognition Moiety

One end of a BacPROTAC uses a target-protein ligand to determine substrate selectivity. For example, JQ1 can serve as a recognition ligand for model BET bromodomain proteins. Its function is to recruit a specific protein into the induced-proximity system rather than directly mediate proteolysis.

(2) ClpC/ClpC1-Recruiting Moiety

The other end is linked to a ClpC- or ClpC1-binding group, allowing the target protein, BacPROTAC, and Clp ATPase to form a ternary complex. ClpC/ClpC1 then captures the target protein, performs ATP-dependent unfolding, and translocates the resulting polypeptide chain into the ClpP or P1P2 proteolytic core without requiring the E3 recruitment or polyubiquitination steps used in classical PROTACs.

(3) Linker Geometric Effects

Linker length, flexibility, and attachment site alter the relative positions of the POI and ClpC/ClpC1. A Linker that is too short may prevent simultaneous binding at both ends, whereas one that is too long may reduce effective proximity. The relative orientation of the POI can also influence whether it enters the AAA+ ATPase substrate-processing pathway. Therefore, BacPROTACs also require evaluation of the complete ternary-complex architecture rather than relying only on the independent Kd values of the two ligands.

5.4 Application of ClpC1 Small-Molecule Ligands

pArg is suitable for mechanistic validation but has limitations in chemical stability and intracellular application. Subsequent BacPROTAC designs can use small-molecule recruiting groups that stably bind ClpC/ClpC1 and connect them with POI ligands such as JQ1 to exploit the ClpC1P1P2 system for processing model substrates. Some ClpC/ClpC1 ligands can also affect AAA+ ATPase oligomeric states and functional activity, meaning that their effects may simultaneously involve substrate redirection and regulation of protease-machine state.

5.5 Mechanistic Comparison Between BacPROTAC and Classical PROTAC

(1) Shared Principle of Induced Proximity

Both technologies use bifunctional molecules to establish a new recognition relationship between a target protein and an endogenous degradation system, thereby altering the substrate spectrum of that degradation machinery.

(2) Different Execution Pathways

Classical PROTAC: POI → PROTAC → E3 ligase → ubiquitination → 26S proteasome.

BacPROTAC: POI → BacPROTAC → ClpC/ClpC1 → ATP-dependent unfolding → ClpP/P1P2 proteolysis.

BacPROTAC represents the transfer of induced-proximity targeted-degradation principles to prokaryotic protein quality-control systems rather than a simple replacement of the E3 ligand in classical PROTACs.

6 Selection of Degradation Platforms for Different Targets

6.1 Target Location and Substrate Scale

6.2 Ligand Availability Is Not the Only Determining Factor

(1) PROTAC

In addition to a POI ligand, the target cell must express an appropriate E3 ligase and form a ternary-complex interface favorable for ubiquitin transfer. If the target protein lacks a suitable geometry for E3 proximity, increasing POI-ligand affinity may still fail to substantially improve degradation.

(2) LYTAC

LYTAC must simultaneously achieve target binding, receptor binding, efficient internalization, and lysosomal sorting. Receptor type, endocytic rate, intracellular trafficking, and receptor recycling all influence final target-protein clearance efficiency.

(3) ATTEC and AUTAC

The target must be able to enter the autophagosome, and the cell must possess intact autophagic flux and lysosomal degradation capacity. If autophagosome formation is normal but terminal lysosomal function is impaired, increasing target recruitment efficiency may still cause accumulation of intermediates rather than effective clearance.

(4) BacPROTAC

In addition to ligands for the target protein and ClpC/ClpC1, bacterial-envelope permeability, efflux systems, Clp protease expression, and whether the target protein can be efficiently captured and unfolded by the AAA+ ATPase must also be considered. Formation of a ternary complex in vitro cannot be directly equated with effective degradation inside bacterial cells.

7 Mechanistic Boundaries of Molecular Glues and RIBOTACs

7.1 Molecular Glues Are Not Equivalent to Non-UPS Degraders

(1) Structural Form

Molecular glues generally lack the typical “POI ligand–Linker–E3 ligand” bifunctional architecture and instead use relatively compact small molecules to induce or stabilize new protein–protein interactions, thereby altering native protein-recognition relationships.

(2) UPS Dependence of Degrading Molecular Glues

Thalidomide, Pomalidomide, and various CELMoDs can bind CRBN and remodel the substrate-recognition interface of CRL4CRBN, causing new substrates to become ubiquitinated and enter the proteasome. Therefore, “molecular glue” defines a molecular mode of inducing protein interactions rather than the terminal degradation pathway and cannot be uniformly classified as a non-ubiquitin–proteasome degradation system.

7.2 RIBOTAC Extends Targeted Degradation to RNA

RIBOTAC (ribonuclease-targeting chimera) uses one end to recognize a specific RNA structure and the other end to recruit an endogenous ribonuclease, thereby inducing selective cleavage of the target RNA. Its common basis with PROTAC lies in selective target recognition and redirection of an endogenous clearance system rather than use of the same substrate or degradation route. It therefore represents extension of induced-degradation strategies from proteins to RNA targets.

8 Experimental Determination of Targeted Degradation Mechanisms

8.1 Evidence Boundaries for Reduced Target Abundance

(1) Exclusion of Expression-Level Changes

A decrease in target protein detected by Western blot only demonstrates a change in protein abundance. Transcriptional inhibition, translational inhibition, cell death, and changes in protein secretion can produce similar results. mRNA levels, cell viability, and time-course analyses can be combined to distinguish active protein degradation from reduced expression.

(2) Confirmation of Degradation-System Dependence

Targeted degradation requires further demonstration that the expected clearance system is involved and that target depletion can be rescued by blocking that system. The most discriminating evidence for different platforms is not simply a reduction in target protein but its corresponding dependence on E3 ligases, lysosomes, autophagy, or Clp proteases.

8.2 PROTAC Validation

(1) Direct Degradation Readouts

Concentration–degradation and time–degradation curves should be established to evaluate parameters such as Dmax, and a sufficiently broad concentration range should be covered to identify a potential hook effect.

(2) UPS Mechanism Validation

Target-protein ubiquitination, knockdown/knockout of the relevant E3 ligase, and proteasome-blockade experiments can be combined. A substantial reduction in degradation after E3 loss or proteasome inhibition supports a UPS-dependent mechanism.

8.3 LYTAC Validation

(1) Distinguishing Internalization from Degradation

Cell-surface target and total target protein should be measured separately. A reduction in surface level while total protein remains stable more strongly supports internalization and redistribution, whereas sustained reduction in total protein is more consistent with actual degradation.

(2) Confirmation of Lysosomal Dependence

Lysosomal-acidification inhibition, lysosomal-function perturbation, and spatial colocalization of the target with markers such as LAMP1/LAMP2 can be combined to determine whether the target enters the terminal lysosomal degradation compartment.

8.4 ATTEC and AUTAC Validation

(1) ATTEC

Target reduction, target–LC3 proximity, autophagic flux, and rescue after autophagy/lysosome blockade should be evaluated together. An increase in LC3-II alone is insufficient evidence because it can result from either enhanced autophagy or blocked autophagic flux.

(2) AUTAC

Target clearance, selective autophagy, and lysosomal dependence should be evaluated. In systems involving K63-linked ubiquitin, the corresponding ubiquitin signal can be further examined. For organelles such as mitochondria, organelle mass or number should also be measured to avoid misinterpreting functional abnormalities such as reduced membrane potential as actual clearance.

8.5 BacPROTAC Validation

BacPROTAC should simultaneously validate target-protein reduction, ClpC/ClpC1 dependence, and dependence on the corresponding protease core. Reconstituted in vitro systems can directly evaluate whether BacPROTAC promotes POI hydrolysis by the Clp system, whereas intracellular studies can use Clp-deficient or Clp-impaired models to determine whether target-protein reduction is correspondingly weakened, thereby distinguishing Clp-dependent protein degradation from direct functional inhibition by the POI ligand and nonspecific cytotoxicity.

9 Products Related to Targeted Degradation, Pathway Validation, and Molecular Construction

9.1 AUTAC- and Autophagy–Lysosome-Related Products

9.2 BacPROTAC Target Ligands and UPS Mechanism-Control Products

9.3 PROTAC Construction Modules and Classical Degradation-Mechanism Control Products

LYTAC, ATTEC, AUTAC, and BacPROTAC respectively extend the applicability boundaries of classical PROTACs in terms of target spatial location, substrate scale, and biological system. Selection among these platforms depends on the biological location and structural characteristics of the target as well as its compatibility with the corresponding endogenous clearance system.

References

[1] Fu Y, Lu B. Targeting lipid droplets for autophagic degradation by ATTEC. Autophagy. 2021;17(12):4486-4488.

[2] Morreale FE, Kleine S, Leodolter J, et al. BacPROTACs mediate targeted protein degradation in bacteria. Cell. 2022;185(13):2338-2353.e18.

[3] Schreiber SL. The Rise of Molecular Glues. Cell. 2021;184(1):3-9.

[4] Li Z, Wang C, Wang Z, et al. Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature. 2019;575(7781):203-209.

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