From the Cradle to the grave: molecular chaperones that may choose between folding and degradation By: Erica Zakhem

Proteins The function of a protein is determined by the 3D structure of the Amino Acid chain The function of a protein is determined by the 3D structure of the Amino Acid chain This 3D structure is produced after translation This 3D structure is produced after translation Proteins are constantly under threat of unfolding due to chemical and cellular stress Proteins are constantly under threat of unfolding due to chemical and cellular stress Hydrogen bonds may be disrupted by change in temperature and varying pH levels Hydrogen bonds may be disrupted by change in temperature and varying pH levels

Protein structure Protein structure and quality is mediated by two systems Molecular chaperones Molecular chaperones Energy-dependent proteases Energy-dependent proteases

Molecular chaperones They aid in the binding of non-native proteins, and inhibit protein aggregation They aid in the binding of non-native proteins, and inhibit protein aggregation Work with regulatory co-chaperones to facilitate the protein folding process Work with regulatory co-chaperones to facilitate the protein folding process

Energy-dependent proteases Eliminate proteins that do not fold according to their native 3D structure Eliminate proteins that do not fold according to their native 3D structure Misfolded proteins are sent to the cell’s degradation machinery for destruction Misfolded proteins are sent to the cell’s degradation machinery for destruction

Hsp70 and Hsp90 The major chaperones found in mammals are the Heat shock proteins 70 and 90 The major chaperones found in mammals are the Heat shock proteins 70 and 90 Hsp70 is involved in the folding of newly synthesized proteins, and in the protection of proteins during cellular stress and protein trafficking Hsp70 is involved in the folding of newly synthesized proteins, and in the protection of proteins during cellular stress and protein trafficking Hsp90 functioning is restricted, but it plays a role in stress protection Hsp90 functioning is restricted, but it plays a role in stress protection

Both these types of chaperones are associated with non-native protein substrates through the hydrophobic portions in the native 3D structure Both these types of chaperones are associated with non-native protein substrates through the hydrophobic portions in the native 3D structure They control conformational regulation of proteins involved in signal transduction, cell proliferation, and apoptosis They control conformational regulation of proteins involved in signal transduction, cell proliferation, and apoptosis They cooperate with the degradation machinery in the cell They cooperate with the degradation machinery in the cell Hsp70 and Hsp90 have been shown to require several co-chaperones in order to act Hsp70 and Hsp90 have been shown to require several co-chaperones in order to act

Co-chaperones They can take either one of two options when regulating chaperone functioning They can take either one of two options when regulating chaperone functioning They can regulate the ATPase cycle of the chaperone, thus influencing its affinity for the protein substrates They can regulate the ATPase cycle of the chaperone, thus influencing its affinity for the protein substrates Or they can recruit the chaperones to specific proteins or protein complexes Or they can recruit the chaperones to specific proteins or protein complexes Many co-chaperones exhibit both chaperone- binding and chaperone-regulating motifs Many co-chaperones exhibit both chaperone- binding and chaperone-regulating motifs

Chaperone-binding motifs Chaperone-binding motifs found in Hsp70 and Hsp90 co-chaperones consist of a tandem arrangement of three degenerate 34 amino acid repeats-tetratricopeptide repeats, TPRs Chaperone-binding motifs found in Hsp70 and Hsp90 co-chaperones consist of a tandem arrangement of three degenerate 34 amino acid repeats-tetratricopeptide repeats, TPRs The Hsp70/Hsp90 organizing protein Hop contains multiple TPRs which allow it to bind to Hsp70 and Hsp90, and to promote the regulation of signal transduction pathways The Hsp70/Hsp90 organizing protein Hop contains multiple TPRs which allow it to bind to Hsp70 and Hsp90, and to promote the regulation of signal transduction pathways

Ubiquitin/proteosome system Major degradation pathway in eukaryotic cells Major degradation pathway in eukaryotic cells Proteins that are to be degraded are labeled with a multi-ubiquitin chain Proteins that are to be degraded are labeled with a multi-ubiquitin chain The proteins are then taken to 26S proteosome which consists of proteases which degrade the proteins. The proteins are then taken to 26S proteosome which consists of proteases which degrade the proteins. Ubiquitylation is mediated by an enzyme complex comprising of E1, E2 and E3 Ubiquitylation is mediated by an enzyme complex comprising of E1, E2 and E3

Carboxyl terminus of Hsp70 Interacting Protein -CHIP CHIP is a co-chaperone that interacts with Hsc70, and it accelerates ubiquitin-dependent degradation of chaperone substrates CHIP is a co-chaperone that interacts with Hsc70, and it accelerates ubiquitin-dependent degradation of chaperone substrates The N-terminal contains three tandem TPRs adjacent to a highly charged α-helix which form a chaperone adaptor The N-terminal contains three tandem TPRs adjacent to a highly charged α-helix which form a chaperone adaptor The C-terminal contains a U-box comprising of a ubiquitin activator (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase enzyme (E3) The C-terminal contains a U-box comprising of a ubiquitin activator (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase enzyme (E3)

The structure of CHIP enables the co- chaperone to link molecular chaperones to the degradation machinery The structure of CHIP enables the co- chaperone to link molecular chaperones to the degradation machinery CHIP targets substrates to the ubiquitin/proteasome system and controls the balance between protein folding and protein degradation CHIP targets substrates to the ubiquitin/proteasome system and controls the balance between protein folding and protein degradation

CHIP plays an integral role in the ubiqutin ligase complex CHIP plays an integral role in the ubiqutin ligase complex Its ability to recognize non-native proteins and specific proteins allows it to select substrates for CHIP-mediated ubiquitylation Its ability to recognize non-native proteins and specific proteins allows it to select substrates for CHIP-mediated ubiquitylation Association with Hsp70 and Hsp90 promotes the ubiquitylation process of various substrates Association with Hsp70 and Hsp90 promotes the ubiquitylation process of various substrates

Chaperone machines Upon association with CHIP chaperones Hsp70 and Hsp90 are turned into degradation factors Upon association with CHIP chaperones Hsp70 and Hsp90 are turned into degradation factors Regulation of protein quality occurs when CHIP occupies co-chaperone sites on Hsp70 and Hsp90 it competes with other co-chaperones Regulation of protein quality occurs when CHIP occupies co-chaperone sites on Hsp70 and Hsp90 it competes with other co-chaperones Hop is a co-chaperone that is a CHIP antagonist Hop is a co-chaperone that is a CHIP antagonist Like CHIP, Hop associates with Hsp70 via the TPR adapter Like CHIP, Hop associates with Hsp70 via the TPR adapter

As opposed to CHIP, Hop assists in chaperone-mediated protein folding As opposed to CHIP, Hop assists in chaperone-mediated protein folding Competing co- chaperones determine whether a chaperone machine is involved in protein folding or protein degradation Competing co- chaperones determine whether a chaperone machine is involved in protein folding or protein degradation

BAG-1 The Hsp70 co-chaperone BAG-1 acts as a link between molecular chaperones and the ubiquitin/proteasome system The Hsp70 co-chaperone BAG-1 acts as a link between molecular chaperones and the ubiquitin/proteasome system This co-chaperone uses its ubiquitin domain for binding to the proteasome, which promotes association between Hsp70 and the proteolytic complex This co-chaperone uses its ubiquitin domain for binding to the proteasome, which promotes association between Hsp70 and the proteolytic complex Increased BAG-1 levels is insufficient to promote degradation Increased BAG-1 levels is insufficient to promote degradation

A co-chaperone complex that includes both CHIP and BAG-1 has been shown to regulate proteasomal sorting of chaperone substrates A co-chaperone complex that includes both CHIP and BAG-1 has been shown to regulate proteasomal sorting of chaperone substrates CHIP binds to the C-terminal while BAG-1 binds to the N-terminal of the chaperone CHIP binds to the C-terminal while BAG-1 binds to the N-terminal of the chaperone BAG-1 competes with Hip, which is a folding- stimulating co0chaperone in the binding to the ATPase of Hsp70 BAG-1 competes with Hip, which is a folding- stimulating co0chaperone in the binding to the ATPase of Hsp70 Again, this illustrates that competing factors determine the function of molecular chaperones Again, this illustrates that competing factors determine the function of molecular chaperones

Hip and BAG-1 compete for binding to the ATPase, while CHIP and Hop compete for association with the C-terminal

Conclusions Hsp70 and Hsp90 interact with many co- chaperones that determine their fate Hsp70 and Hsp90 interact with many co- chaperones that determine their fate Co-chaperones have been used elucidate the roles of chaperones in protein folding and degradation Co-chaperones have been used elucidate the roles of chaperones in protein folding and degradation Our knowledge of chaperones and co- chaperone mechanisms are still limited Our knowledge of chaperones and co- chaperone mechanisms are still limited