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The modern biopharmaceutical industry traces its roots to the dawn of the twentieth century, coincident with marketing of aspirin—a signature event in the history of modern drug development. Although the archetypal discovery process did not change markedly in the first seven decades of the industry, the past fifty years have seen two successive waves of transformative innovation in the development of drug molecules: the rise of ‘rational drug discovery’ methodology in the 1970s, followed by the invention of recombinant protein-based therapeutic agents in the 1980s. An incipient fourth wave is the advent of multispecific drugs. The successful development of prospectively designed multispecific drugs has the potential to reconfigure our ideas of how target-based therapeutic molecules can work, and what it is possible to achieve with them. Here I review the two major classes of multispecific drugs: those that enrich a therapeutic agent at a particular site of action and those that link a therapeutic target to a biological effector. The latter class—being freed from the constraint of having to directly modulate the target upon binding—may enable access to components of the proteome that currently cannot be targeted by drugs. The development and future prospects of prospectively designed multispecific drugs, which have the potential to transform the biopharmaceutical industry by enabling the targeting of currently inaccessible components of the proteome, are reviewed.

Genomic alterations may make cancer cells more dependent than normal cells on mechanisms of proteostasis, including protein folding and degradation. This proposition is the basis for the clinical use of proteasome inhibitors to treat multiple myeloma and mantle cell lymphoma. However, proteasome inhibitors have not proved effective in treating other cancers, and this has called into question the general applicability of this approach. Here, I consider possible explanations for this apparently limited applicability, and discuss whether inhibiting other broadly acting components of the ubiquitin-proteasome system - including ubiquitin-activating enzyme and the AAA-ATPase p97/VCP - might be more generally effective in cancer therapy.

Key Points The cullin–RING ligases (CRLs) comprise a superfamily of ubiquitin ligases that are implicated in the regulation of a diverse array of eukaryotic functions. The various cullin proteins function as a rigid scaffold for the assembly of this modular class of ligase. All cullins associate with a RING protein through their C-terminal domain, whereas the N-terminal region recruits a wide variety of receptor proteins that confer substrate specificity. The cullin–RING module is often referred to as the catalytic core, because it is common to all CRLs. It recruits ubiquitin-conjugating enzymes (E2s) and activates the transfer of ubiquitin from the E2 to the substrate through an as-yet-unclear mechanism that does not involve a CRL–ubiquitin-thioester intermediate. The substrate receptors for CRLs are generally linked to the catalytic core through adaptor proteins that are specific for each cullin-family member. Numerous substrate receptors can be recruited to each CRL core, which increases the diversity of proteins that can be targeted for ubiquitylation. In most cases, the recognition of a substrate by a CRL requires post-translational modification of the substrate. This further increases the repertoire of substrates that can be targeted to a given CRL and also links protein ubiquitylation and turnover to numerous signalling pathways. CRL activity can be regulated by numerous mechanisms, which include the turnover of substrate receptors, the reversible attachment of the ubiquitin-like protein NEDD8 to cullins, and the sequestration of cullins by CAND1. Cullin–RING complexes comprise the largest known class of ubiquitin ligases. Owing to the great diversity of their substrate-receptor subunits, it is possible that there are hundreds of distinct cullin–RING ubiquitin ligases in eukaryotic cells, which establishes these enzymes as key mediators of post-translational protein regulation. In this review, we focus on the composition, regulation and function of cullin–RING ligases, and describe how these enzymes can be characterized by a set of general principles.

p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo–electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)–bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5′-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., ‘bounce-back’) of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. Cells exposed to high temperatures, infections and other forms of stress often produce oxygen ions and peroxide molecules that can cause damage to proteins and DNA. Cells therefore rely on molecular machines called proteasomes to eliminate damaged proteins, before they cause too much harm. Two related transcription factors—proteins that interact with DNA to ‘switch on’ the expression of genes—are involved in a cell’s responses to stress, but in different ways. Nrf2 switches on genes that limit the damage caused by oxygen ions and peroxide molecules, while Nrf1 switches on the genes that encode the components of the proteasome. As such, Nrf1 helps to restart proteasome activity if it has been shut off—a phenomenon known as ‘bounce-back’. Within a cell, Nrf1 is known to start off embedded within the membranes of a structure called the endoplasmic reticulum. However, it is not clear how activated Nrf1 leaves this membrane and enters the nucleus to interact with the cell’s DNA. Now, Radhakrishnan et al. show that when Nrf1 is produced, most of its length is found inside the endoplasmic reticulum, with only a small piece being anchored in the surrounding membrane. This is unlike previously described transcription factors that associate with the endoplasmic reticulum, which are stuck to the outside of this structure. Radhakrishnan et al. also discovered that the activation of Nrf1 depends on an enzyme called p97 or VCP. This enzyme helps to flip Nrf1 from the inside of the endoplasmic reticulum to its outside surface. In most cells, the proteasome then breaks down this part of Nrf1. However, if the proteasome is inhibited, an unknown enzyme cuts Nrf1 free from the endoplasmic reticulum, allowing it to migrate to the nucleus and promote the production of more proteasome components to counteract the inhibition. Interestingly, drugs that inhibit the proteasome are used to combat cancer because the build-up of damaged proteins is toxic to the cancer cells. By showing that p97 promotes the ‘bounce-back’ of the proteasome, the work of Radhakrishnan et al. suggests that combining existing proteasome inhibitors with drugs that inhibit p97 could eventually lead to new, more effective, therapies for cancer or other diseases.

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin–proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness. Proteasomal overload can be found in a broad spectrum of mouse models of retinal degeneration. Here the authors find that overexpressing the PA28α subunit of the 11S proteasome cap increased the number of surviving functional photoreceptor cells in a mouse model of retinal degeneration bearing the P23H mutation in rhodopsin.

Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97. Ribosomes are complex molecular machines that translate the sequence of bases in a messenger RNA (mRNA) transcript into a polypeptide that subsequently folds to form a protein. Each ribosome is composed of two major subunits: the small subunit reads the mRNA transcript, and the large subunit joins amino acids together to form the polypeptide. This process stops when the ribosome encounters a stop codon and releases the completed polypeptide. It is critical that cells perform some form of quality control on the polypeptides as they are translated to prevent a build up of incomplete, incorrect or toxic proteins in cells. Problems can occur if a ribosome stalls while translating the mRNA transcript, or if the mRNA transcript is defective. For example, most mRNA transcripts contain a stop codon, but some do not, and these non-stop mRNA transcripts result in a non-stop polypeptide that remains tethered to the ribosome. It is important that the cell identifies and removes these faulty polypeptides so as to leave the ribosome free to translate other (non-faulty) mRNA transcripts. A regulatory protein called ubiquitin is responsible for marking and sending proteins that are faulty, or are no longer needed by the cell, to a molecular machine called the proteasome, where they are degraded by a process called proteolysis. In 2010 researchers identified Ltn1 as the enzyme that attaches ubiquitin to non-stop proteins in yeast. Now, building on this work, Verma et al. identify additional proteins involved in this process. In particular, an ATPase enzyme called Cdc48 (known as p97 or VCP in human cells) and two co-factors—Ufd1 and Npl4—promote release of the ubiquitinated non-stop polypeptides from the ribosomes, thus committing the marked polypeptide to destruction by the proteasome. Verma et al. also show that the Cdc48-Ufd1-Npl4 complex is involved in other aspects of quality control of newly synthesized proteins within cells. Collectively these processes are known as ribosome-associated degradation. Mutations of the gene that codes for human p97 can cause a number of diseases, including Paget's disease of the bone and frontotemporal dementia, so an improved understanding of ribosome-associated degradation could provide new insights into these diseases.

PROTACs are heterobifunctional small molecules that simultaneously bind a target protein and a ubiquitin ligase, enabling ubiquitination and degradation of the target. Major progress in developing potent and specific PROTACs has recently been reported, invigorating prospects for novel PROTAC-based therapies.

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. Ribosomes are the molecular machines in cells that produce proteins. The ribosomes themselves are composed of almost 80 different proteins that are held together by scaffolds made from molecules of RNA. Each protein is present in one copy, and so equal numbers of all proteins are needed to assemble a ribosome. However, because it takes many steps to produce a protein and biological processes are inherently imprecise, it is essentially impossible for a cell to produce exactly the same number of copies of all the proteins in a ribosome. Much research suggests that, to overcome these issues, a cell will make more of certain ribosomal proteins than it needs, and then degrade the leftovers that are not used. However, it was not clear how this happens, nor was it known what are the consequences of failing to degrade the leftovers. Now, Sung et al. show that yeast cells use an enzyme named Tom1 to attach a protein-marker called ubiquitin to ribosomal proteins that are made in excess and not assembled into ribosomes. The ubiquitin serves as a tag that marks proteins for degradation, and yeast cell that lack Tom1 fail to degrade any excess ribosomal proteins. Consequently, the mutant yeast become sensitive to any factors that alter the balance of the protein and RNA building blocks used to assemble ribosomes. The human equivalent of Tom1 is known as Huwe1, and the data of Sung et al. suggest that this enzyme acts in a similar pathway. Further experiments are now needed to explore the role of Huwe1 in greater depth, and investigate if problems with this enzyme are associated with any human diseases. Finally, working out the exactly how Tom1 recognizes unassembled ribosomal proteins will be another important challenge for future studies.

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4CRBN and p97 pathways.