A genome-wide synthetic dosage lethality screen reveals multiple pathways that require the functioning of ubiquitin-binding proteins Rad23 and Dsk2

Background  

Ubiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. A classic role for ubiquitin is to flag proteins for destruction by the proteasome. Recent studies indicate that ubiquitin-binding proteins (e.g. Rad23, Dsk2, Rpn10) play a pivotal role in transferring ubiquitylated proteins to the proteasome. However, the specific role of these ubiquitin receptors remains poorly defined. A key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. Although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging.     

Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response

The ordered assembly of DNA repair factors on chromatin has been studied in great detail, whereas we are only beginning to realize that selective extraction of proteins from chromatin plays a central role in the DNA damage response. Interestingly, the protein modifier ubiquitin not only regulates the well-documented recruitment of repair proteins, but also governs the temporally and spatially controlled extraction of proteins from DNA lesions. The facilitator of protein extraction is the ubiquitin-dependent ATPase valosin-containing protein (VCP)/p97 complex, which, through its segregase activity, directly extracts ubiquitylated proteins from chromatin. In this review, we summarize recent studies that uncovered this important role of VCP/p97 in the cellular response to genomic insults and discuss how ubiquitin regulates two intuitively counteracting activities at sites of DNA damage.     

The ubiquitin proteoform problem

The diversity of ubiquitin modifications is immense. A protein can be monoubiquitylated, multi-monoubiquitylated, and polyubiquitylated with chains varying in size and shape. Ubiquitin itself can be adorned with other ubiquitin-like proteins and smaller functional groups. Considering different combinations of post-translational modifications can give rise to distinct biological outcomes, characterizing ubiquitylated proteoforms of a given protein is paramount. In this Opinion, we review recent advances in detecting and quantifying various ubiquitin proteoforms using mass spectrometry.     

Ubiquitylation of an ERAD substrate occurs on multiple types of amino acids

Any protein synthesized in the secretory pathway has the potential to misfold and would need to be recognized and ubiquitylated for degradation. This is astounding, since only a few ERAD-specific E3 ligases have been identified. To begin to understand substrate recognition, we wished to map the ubiquitylation sites on the NS-1 nonsecreted immunoglobulin light chain, which is an ERAD substrate. Ubiquitin is usually attached to lysine residues and less frequently to the N terminus of proteins. In addition, several viral E3s have been identified that attach ubiquitin to cysteine or serine/threonine residues. Mutation of lysines, serines, and threonines in the NS-1 variable region was necessary to significantly reduce ubiquitylation and stabilize the protein. The Hrd1 E3 ligase was required to modify all three amino acids. Our studies argue that ubiquitylation of ER proteins relies on very different mechanisms of recognition and modification than those used to regulate biological processes.     

IAPS and ubiquitylation

The Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death, whose amplification has been correlated with tumor progression. Due to the presence of a RING domain, IAP proteins are classed as ubiquitin ligases and regulate cell survival by orchestrating a variety of ubiquitin modifications. Ubiquitin protein modification is fundamental in cell signaling and different ubiquitin modifications may label proteins for destruction, relocalization or provide a recruitment platform for ubiquitin binding proteins. Ubiquitin performs a myriad of different functions because it can be conjugated to a large range of target proteins through numerous different types of ubiquitin linkages. Despite the fact that ubiquitin is extremely versatile, the E3s such as the IAPs provide an important level of control due to their specificity for certain substrates. Several recent reviews have discussed the role of IAPs in regulating immune signaling so we have therefore focused our review on the interplay between IAPs and ubiquitin and discussed the importance of this relationship for the regulation of themselves, specific substrates and various cell death and survival signaling pathways.     

When the chains do not break: the role of USP10 in physiology and pathology

Deubiquitination is now understood to be as important as its partner ubiquitination for the maintenance of protein half-life, activity, and localization under both normal and pathological conditions. The enzymes that remove ubiquitin from target proteins are called deubiquitinases (DUBs) and they regulate a plethora of cellular processes. DUBs are essential enzymes that maintain intracellular protein homeostasis by recycling ubiquitin. Ubiquitination is a post-translational modification where ubiquitin molecules are added to proteins thus influencing activation, localization, and complex formation. Ubiquitin also acts as a tag for protein degradation, especially by proteasomal or lysosomal degradation systems. With ~100 members, DUBs are a large enzyme family; the ubiquitin-specific peptidases (USPs) being the largest group. USP10, an important member of this family, has enormous significance in diverse cellular processes and many human diseases. In this review, we discuss recent studies that define the roles of USP10 in maintaining cellular function, its involvement in human pathologies, and the molecular mechanisms underlying its association with cancer and neurodegenerative diseases. We also discuss efforts to modulate USPs as therapy in these diseases.Subject terms: Cell biology, Cell signalling     

Illuminating the ubiquitin/proteasome system

The ubiquitin/proteasome system (UPS) is responsible for the regulated processive degradation of proteins residing in the cytosol, nucleus, and endoplasmic reticulum. The two central players are ubiquitin, a small protein that is conjugated to substrates, and the proteasome, a large multi-subunit proteolytic complex that executes degradation of ubiquitylated proteins. Ubiquitylation and proteasomal degradation are highly dynamic processes. During the last decade, many researchers have started taking advantage of fluorescent proteins, which allow studying the dynamic nature of this system in the context of its natural environment: the living cell. In this review, we will summarize studies that have implemented this approach to examine the UPS and discuss novel insights in the dynamic organization of the UPS.     

Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting

Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.     

Hul5 ubiquitin ligase

Failure to eliminate abnormal proteins in the cell is associated with numerous aggregation diseases. Misfolded proteins are normally detected by protein quality control and either refolded or eliminated. The ubiquitin-proteasome system is a major pathway that degrades these unwanted proteins. Ubiquitin ligases are central to these degradation pathways as they recognize aberrant proteins and covalently attach a polyubiquitin chain to target them to the proteasome. We discovered that the Hul5 ubiquitin ligase is a major player in a novel protein quality control pathway that targets cytosolic misfolded proteins. Hul5 is required for the maintenance of cell fitness and the increased ubiquitination of low solubility proteins after heat-shock in yeast cells. We identified several low-solubility substrates of Hul5, including the prion-like protein Pin3. It is now apparent that in the cytoplasm, misfolded proteins can be targeted by multiple degradation pathways. In this review, we discuss how the Hul5 protein quality control pathway may specifically target low solubility cytosolic proteins in the cell.     

Hul5 ubiquitin ligase: Good riddance to bad proteins

Failure to eliminate abnormal proteins in the cell is associated with numerous aggregation diseases. Misfolded proteins are normally detected by protein quality control and either refolded or eliminated. The ubiquitin-proteasome system is a major pathway that degrades these unwanted proteins. Ubiquitin ligases are central to these degradation pathways as they recognize aberrant proteins and covalently attach a polyubiquitin chain to target them to the proteasome. We discovered that the Hul5 ubiquitin ligase is a major player in a novel protein quality control pathway that targets cytosolic misfolded proteins. Hul5 is required for the maintenance of cell fitness and the increased ubiquitination of low solubility proteins after heat-shock in yeast cells. We identified several low-solubility substrates of Hul5, including the prion-like protein Pin3. It is now apparent that in the cytoplasm, misfolded proteins can be targeted by multiple degradation pathways. In this review, we discuss how the Hul5 protein quality control pathway may specifically target low solubility cytosolic proteins in the cell.     

Ubiquitin: not just for proteasomes anymore

Ubiquitin is a small protein that can be covalently linked to itself or other proteins, either as single ubiquitin molecules or as chains of polyubiquitin. Addition of ubiquitin to a target protein requires a series of enzymatic activities (by ubiquitin-activating, -conjugating and -ligating enzymes). The first function attributed to ubiquitin was the covalent modification of misfolded cytoplasmic proteins, thereby directing proteasome-dependent proteolysis. More recently, additional functions have been ascribed to ubiquitin and ubiquitin-related proteins. Ubiquitin directs specific proteins through the endocytic pathway by modifying cargo proteins, and possibly also components of the cytoplasmic protein trafficking machinery.     

Ubiquitin interactions of NZF zinc fingers

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.     

Ubiquitin receptor proteins hHR23a and hPLIC2 interact

Ubiquitin receptor proteins play an important role in delivering ubiquitylated protein substrates to the proteasome for degradation. HHR23a and hPLIC2 are two such ubiquitin receptors that contain ubiquitin-like (UBL) domains, which interact with the proteasome, and ubiquitin-associated (UBA) domains, which interact with ubiquitin. Depending on their abundance UBL/UBA family members can either promote or inhibit the degradation of other proteins, which suggests their participation in the delivery of substrates to the proteasome is highly regulated. In previous work, we determined UBL/UBA domain interactions to promote intramolecular interactions in hHR23a that are abrogated with the addition of either ubiquitin or the proteasome component S5a. In yeast, we determined the hHR23a ortholog (Rad23) to interact with another UBL/UBA family member (Ddi1) and to bind a common tetraubiquitin chain. Here, we use NMR spectroscopy to reveal that hHR23a interacts with hPLIC2 via UBL/UBA domain interactions and to map their binding surfaces. In addition, we demonstrate that these two proteins associate in mammalian cells. Intriguingly, inhibition of the proteasome mitigates hHR23a/hPLIC2 interaction.     

ULK1 ubiquitylation is regulated by phosphorylation on its carboxy terminus

Autophagy is a highly conserved process that acts sequestering cytoplasmic components for their degradation by the lysosomes. It consists of several sequential steps that have to be finely regulated to ensure both its progression and termination. Post-translational modifications (PTMs) play an important role in regulating ATG proteins function in different stages of autophagy. Recently, we demonstrated that, during prolonged starvation, ULK1 protein is specifically ubiquitylated by NEDD4L, and that this regulation is important to protect cells against excessive autophagy. In this Extra view, we show that ULK1 phosphorylation at 3 different sites on the same ULK1 target region for NEDD4L is preparatory for its ubiquitylation and subsequent degradation. This adds to the complexity of ULK1 multi-level regulation by several factors, including kinases, phosphatases and acetylases, with each contributing to autophagy homeostasis     

A superfamily of protein tags: ubiquitin,SUMO and related modifiers

The biological functions of many proteins are altered by their covalent attachment to polypeptide modifiers. The best-known example of this type of modification is ubiquitination. Ubiquitin has a well-documented role in targeting proteins for degradation by the proteasome, but additional effects of protein ubiquitination are now being uncovered. Furthermore, multiple polypeptides that are distinct from, but related to, ubiquitin are also enzymatically coupled to target macromolecules, and these ubiquitin-like proteins participate in diverse biological processes such as DNA repair, autophagy and signal transduction.     

The novel functions of ubiquitination in signaling

Ubiquitin is best known for its function in targeting proteins for degradation by the proteasome. Recent studies have revealed several new functions of ubiquitin that are independent of proteasomal degradation. These functions include the novel signaling roles of ubiquitin in DNA repair and the activation of protein kinases such as IkappaB kinase. In both cases, a novel form of polyubiquitin chain linked through lysine-63 of ubiquitin plays an important regulatory role. Monoubiquitination also has signaling roles that are distinct from those of polyubiquitination, as illustrated from the studies of DNA repair. Thus, polyubiquitination and monoubiquitination have emerged as important signaling mechanisms that control diverse physiological and pathological processes.     

泛素连接酶的结构与功能研究进展

泛素化是体内蛋白质翻译后重要修饰之一,是蛋白质降解的信号.泛素连接酶E3是泛素化过程中的关键酶之一,介导活化的泛素从结合酶E2转移到底物,不同的泛素连接酶作用于不同的底物蛋白,决定了泛素化修饰的特异性.根据结构与功能机制的不同,可将泛素连接酶E3分为HECT (homologousto E6AP C terminus)家族和RING-finger家族,前者含有HECT结构域,可直接与泛素连接再将其传递给底物.RING-finger家族的E3发现较晚,庞大且功能复杂,是近年来研究的热点,此家族均包含相似的E2结合结构域和特异的底物结合部分,作为桥梁将活化的泛素从E2直接转移到靶蛋白,其本身并不与泛素发生作用.总结了这2种E3连接酶家族成员的三维结构及功能机制研究的最新进展.     

泛素蛋白磷酸化修饰的功能与解析

泛素化是存在于真核生物中一种重要的翻译后修饰过程,参与调控包括蛋白质降解在内的多种生命活动。实现这一调控过程需要将一个由76个氨基酸组成的泛素蛋白共价连接到底物蛋白上。同时,泛素本身也存在多种翻译后修饰,包括泛素化、磷酸化、乙酰化等,进一步丰富了泛素的修饰类型,决定了底物蛋白不同的命运。近年来,伴随着第65位丝氨酸磷酸化泛素蛋白参与调控线粒体自噬这一突破性进展,泛素蛋白其余磷酸化位点的功能研究也获得越来越多的关注。本文根据目前已有的国内外研究和报道,总结了泛素蛋白已知的磷酸化修饰位点,梳理了泛素蛋白第12位和66位苏氨酸、第57位和65位丝氨酸等位点的磷酸化修饰对其生物物理特性带来的改变,并对相应修饰位点所涉及的生物学功能调控进行了综述。     

Ubiquitin: structures, functions, mechanisms

Ubiquitin is the founding member of a family of structurally conserved proteins that regulate a host of processes in eukaryotic cells. Ubiquitin and its relatives carry out their functions through covalent attachment to other cellular proteins, thereby changing the stability, localization, or activity of the target protein. This article reviews the basic biochemistry of these protein conjugation reactions, focusing on ubiquitin itself and emphasizing recent insights into mechanism and specificity.