Subunit S5a Research Articles

Mapping the Interactions between Lys48 and Lys63-Linked Di-ubiquitins and a Ubiquitin-Interacting Motif of S5a

Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as nonproteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.

Actinfilin Is a Cul3 Substrate Adaptor, Linking GluR6 Kainate Receptor Subunits to the Ubiquitin-Proteasome Pathway

Kainate receptors have been implicated in excitotoxic neuronal death induced by diseases such as epilepsy and stroke. Actinfilin, a synaptic member of the BTB-Kelch protein family, is known to bind to the actin cytoskeleton. However, little is understood about its function at the synapse. Here, we report that actinfilin is able to bind to GluR6, a kainate-type glutamate receptor subunit, and target GluR6 for degradation. Like many members of its protein family, actinfilin acts as a substrate adaptor, binding Cullin 3 (Cul3) and linking GluR6 to the E3 ubiquitin-ligase complex. We map this interaction to the Kelch repeat domain of actinfilin and the GluR6 C terminus. Co-immunoprecipitation and immunofluorescence studies show that GluR6 is ubiquitinated, and that GluR6 levels are decreased by actinfilin overexpression but increased when actinfilin levels are reduced by specific RNA interference. Furthermore, actinfilin-Cul3 interactions appear to be important for regulating surface GluR6 expression. Synaptic GluR6 levels are elevated in mice with lowered neuronal Cul3 expression and when dominant-negative forms of Cul3 are transfected into hippocampal neurons. Together our data demonstrate that actinfilin acts as a scaffold, linking GluR6 to the Cul3 ubiquitin ligase to provide a novel mechanism for kainate receptor degradation.

The UBL domain of PLIC‐1 regulates aggresome formation

Defects in protein folding and the proteasomal pathway have been linked with many neurodegenerative diseases. PLIC-1 (protein linking IAP to the cytoskeleton) is a ubiquitin-like protein that binds to the ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a. Here, we show that PLIC-1 also binds to the UIM proteins ataxin 3--a deubiquitinating enzyme--HSJ1a--a co-chaperone--and EPS15 (epidermal growth factor substrate 15)--an endocytic protein. Using a polyglutamine (polyQ) disease model, we found that both endogenous PLIC-1 and EPS15 localize to perinuclear aggresomes, and that polyQ enhances their in vivo interaction. We show that knockdown of PLIC-1 and EPS15 by RNA interference reduces aggresome formation. In addition, PLIC-1(DeltaUBL) functions as a dominant-negative mutant, blocking both polyQ transport to aggresomes and the association of EPS15 with dispersed aggregates. We also show that PLIC-1 is upregulated by arsenite-induced protein misfolding. These results indicate a role for PLIC-1 in the protein aggregation-stress pathway, and we propose a novel function for the ubiquitin-like (UBL) domain--by means of UBL-UIM interactions--in transport to aggresomes.

A Role for Parkin in Regulating EGFR Endocytosis

Ubiquitination is a posttranslational modification most familiar for its role in targeting proteins for proteasomal degradation. However, Fallon et al. discovered that the ubiquitin-like domain (Ubl) of parkin [an E3 ubiquitin ligase, mutation of which has been implicated in a familial form of Parkinson's disease (PD)] failed to interact with ubiquitin-interacting motifs (UIMs) within the proteasomal S5a subunit, leading them to search for other ubiquitin-dependent roles for parkin. Pulldown assays revealed both Ubl-UIM-mediated and Ubl-independent interactions between parkin and Eps15, an adaptor protein involved in endocytosis of the epidermal growth factor (EGF) receptor (EGFR). EGF treatment of Her14 cells transiently increased association of parkin with Eps15 as well as association of parkin and Eps15 with EGFR. EGF stimulates Eps15 ubiquitination, and this was enhanced by expression of wild-type parkin (which ubiquitinated Eps15 in vitro) but not by two mutant forms associated with PD. Experiments involving parkin overexpression or loss in cell lines indicated that parkin slowed EGFR endocytosis; moreover, lack of parkin was associated with a decrease in EGF-stimulated Akt phosphorylation in both HeLa cells and mouse brain synaptosomes. The effects of parkin on EGFR endocytosis and Akt signaling both depended on the presence of Eps15. Noting that EGFR signaling protects dopaminergic neurons in models of PD, the authors suggest that the loss of parkin-dependent regulation of EGFR endocytosis and Akt signaling (mediated through its ubiquitination of Eps15) may contribute to the pathogenesis of PD. Husnjak and Dikic discuss the research in a News and Views article. L. Fallon, C. M. L. Belanger, A. T. Corera, M. Kontogiannea, E. Regan-Klapisz, F. Moreau, J. Voortman, M. Haber, G. Rouleau, T. Thorarinsdottir, A. Brice, P. M. P. van Bergen en Henegouwen, E. A. Fon, A regulated interaction with the UIM protein Eps15 implicates parkin in EGF receptor trafficking and PI(3)K-Akt signalling. Nat. Cell Biol. 8 , 834-842 (2006). [PubMed] K. Husnjak, I. Dikic, EGFR trafficking: Parkin' in a jam. Nat. Cell Biol. 8 , 787-788 (2006). [PubMed]

Modification of Cul1 regulates its association with proteasomal subunits

BackgroundUbiquitylation targets proteins for degradation by the 26S proteasome. Some yeast and plant ubiquitin ligases, including the highly conserved SCF (Skp1/Cul1/F-box protein) complex, have been shown to associate with proteasomes. We sought to characterize interactions between SCF complexes and proteasomes in mammalian cells.ResultsWe found that the binding of SCF complexes to proteasomes is conserved in higher eukaryotes. The Cul1 subunit associated with both sub-complexes of the proteasome, and high molecular weight forms of Cul1 bound to the 19S proteasome. Cul1 is ubiquitylated in vivo. Ubiquitylation of Cul1 promotes its binding to the S5a subunit of the 19S sub-complex without affecting Cul1 stability.ConclusionThe association of ubiquitylating enzymes with proteasomes may be an additional means to target ubiquitylated substrates for degradation.

Interaction of NUB1 with the proteasome subunit S5a

NUB1 interacts with a ubiquitin-like protein NEDD8 to target the NEDD8 monomer and neddylated proteins to the proteasome for degradation. Therefore, NUB1 is thought to be a potent downregulator of NEDD8 conjugation system. Since NUB1 possesses a UBL domain, which was previously shown to be an S5a-interacting motif in RAD23/HHR23, we initially hypothesized that NUB1 interacts with the S5a subunit of the proteasome through its UBL domain. To examine this, we performed an in vitro GST pull-down assay and a yeast two-hybrid assay. Unexpectedly, our studies revealed that NUB1 directly interacts with the S5a subunit through its C-terminal region between amino acid residues 536 and 584, not through its UBL domain. Although the UBL domain was not an S5a-interacting motif in NUB1, our further studies revealed that the UBL domain is required for the function of NUB1.

Structure of S5a Bound to Monoubiquitin Provides a Model for Polyubiquitin Recognition

Ubiquitin is a key regulatory molecule in diverse cellular events. How cells determine the outcome of ubiquitylation remains unclear; however, a likely determinant is the specificity of ubiquitin receptor proteins for polyubiquitin chains of certain length and linkage. Proteasome subunit S5a contains two ubiquitin-interacting motifs (UIMs) through which it recruits ubiquitylated substrates to the proteasome for their degradation. Here, we report the structure of S5a (196-306) alone and complexed with two monoubiquitin molecules. This construct contains the two UIMs of S5a and we reveal their different ubiquitin-binding mechanisms and provide a rationale for their unique specificities for different ubiquitin-like domains. Furthermore, we provide direct evidence that S5a (196-306) binds either K63-linked or K48-linked polyubiquitin, and in both cases prefers longer chains. On the basis of these results we present a model for how S5a and other ubiquitin-binding proteins recognize polyubiquitin.

Letter to the Editor: Chemical shift assignments of the (poly)ubiquitin-binding region of the proteasome subunit S5a

Biological context Ubiquitin recognition is essential for numerous cellular processes including protein degradation, endocytic sorting, transcriptional control, intranuclear localization, and DNA repair. Indeed the biological impact of ubiquitylation has recently been realized to be comparable to phosphorylation. Ubiquitylation as a signal for targeted protein degradation by the proteasome has been well studied due to its biological prominence. Among the duties of the ubiquitin-proteasome pathway is controlling the lifespan of regulatory proteins, removing improperly folded proteins that would otherwise aggregate, and producing immunocompetent peptides. Indeed failure of proteasome-mediated protein degradation is associated with numerous neurodegenerative diseases, including Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and poly

The Effects of the Polyglutamine Repeat Protein Ataxin-1 on the UbL-UBA Protein A1Up

The ataxin-1 interacting ubiquitin-like protein (A1Up) contains an amino-terminal ubiquitin-like (UbL) region, four stress-inducible, heat shock chaperonin-binding motifs (STI1), and an ubiquitin-associated domain (UBA) at the carboxyl terminus of A1Up. Although proteins that have both an UbL and UBA domain are thought to play a crucial role in proteasome-mediated activities, few are characterized, except for hHR23A/B. Similar to other UbL-containing proteins, the UbL of A1Up is essential for the interaction of A1Up with the S5a subunit of the 19S proteasome. Importantly, the interaction with the 19S proteasome was disrupted in the presence of the polyglutamine repeat protein, ataxin-1. The UbL domain of A1Up is ubiquitinated by both Lys(48)-linked and Lys(63)-linked chains. Intact A1Up is stable, suggesting that ubiquitination of A1Up is important for degradation-independent targeting of A1Up to the 19S proteasome. The UBA domain of A1Up binds polyubiquitin chains and has a role in the stability of A1Up and in the subcellular localization of A1Up. When the UBA domain was deleted, the localization of A1Up was entirely cytoplasmic, and it co-localized with the proteasome. Interestingly, the interaction between A1Up and mutant ataxin-1-(82Q) increased the half-life of A1Up, whereas nonpathogenic wild-type ataxin-1-(30Q) or ataxin-1-(82Q)-A776 did not.

Co-chaperone CHIP associates with mutant Cu/Zn-superoxide dismutase proteins linked to familial amyotrophic lateral sclerosis and promotes their degradation by proteasomes

Although the ubiquitin–proteasome system and the molecular chaperones are implicated to play an important role in pathogenesis of familial amyotrophic lateral sclerosis (FALS) caused by mutations in Cu/Zn-superoxide dismutase (SOD1), the mechanism underlying the causes of this fatal disease is still poorly understood. Here we found that co-chaperone CHIP (carboxyl terminus of Hsc70-interacting protein), together with molecular chaperones Hsc70/Hsp70 and Hsp90, associates with FALS-linked mutant SOD1 proteins in cultured human cells. S5a subunit of 26S proteasomes, which recognizes polyubiquitylated proteins, also interacts with mutant SOD1 proteins. Over-expression of CHIP leads to the reduction in cellular levels of mutant SOD1 as well as the suppression of cytotoxicity induced by mutant SOD1. Unusually, rather than increasing the level of poly-ubiquitylated SOD1, over-expressed CHIP alters the ubiquitylation pattern of mutant SOD1 proteins. Both down-regulation and ubiquitylation of mutant SOD1 are greatly reduced by a mutant CHIP protein lacking U-box domain. Taken together, these results suggest that co-chaperone CHIP, possibly with another E3 ligase(s), modulates the ubiquitylation of mutant SOD1 and renders them more susceptible for proteasomal degradation.

CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70.

Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1(G93A) transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome.

Structure of the Ubiquitin-interacting Motif of S5a Bound to the Ubiquitin-like Domain of HR23B

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves signaling functions that control several cellular processes. The ubiquitination signal is recognized by downstream effectors, many of which carry a ubiquitin-interacting motif (UIM). Such interactions can be modulated by regulators carrying a ubiquitin-like (UbL) domain, which binds UIM by mimicking ubiquitination. Of them, HR23B regulates the proteasomal targeting of ubiquitinated substrates, DNA repair factors, and other proteins. Here we report the structure of the UIM of the proteasome subunit S5a bound to the UbL domain of HR23B. The UbL domain presents one hydrophobic and two polar contact sites for interaction with UIM. The residues in these contact sites are well conserved in ubiquitin, but ubiquitin also presents a histidine at the interface. The pH-dependent protonation of this residue interferes with the access of ubiquitin to the UIM and the ubiquitin-associated domain (UBA), and its mutation to a smaller residue increases the affinity of ubiquitin for UIM.

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a.

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation.

Structural determinants for the binding of ubiquitin-like domains to the proteasome.

HHR23A, a protein implicated in nucleotide excision repair, belongs to a class of proteins containing both a ubiquitin-like (Ubl) domain and one or more ubiquitin-associated (UBA) domains, suggesting a role in the ubiquitin-proteasome pathway as well. The Ubl domain binds with high affinity to the second ubiquitin-interacting motif (UIM) of the S5a subunit of the proteasome. Here we present the solution structures of the HHR23A Ubl domain, the second UIM of S5a (UIM-2), and the Ubl:S5a-UIM-2 complex. The HHR23A Ubl domain is structurally similar to ubiquitin. The S5a UIM forms an alpha-helix with an unexpected hairpin loop that contributes to the binding interface with Ubl. The molecular determinants of the Ubl-proteasome interaction are revealed by analysis of the structures, chemical shift mapping, mutant binding studies and sequence conservation.

Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis.

Machado-Joseph disease is caused by an expansion of a trinucleotide CAG repeat in the gene encoding the protein ataxin-3. We investigated if ataxin-3 was a proteasome-associated factor that recognized ubiquitinated substrates based on the rationale that (i) it is present with proteasome subunits and ubiquitin in cellular inclusions, (ii) it interacts with human Rad23, a protein that may translocate proteolytic substrates to the proteasome, and (iii) it shares regions of sequence similarity with the proteasome subunit S5a, which can recognize multiubiquitinated proteins. We report that ataxin-3 interacts with ubiquitinated proteins, can bind the proteasome, and, when the gene harbors an expanded repeat length, can interfere with the degradation of a well-characterized test substrate. Additionally, ataxin-3 associates with the ubiquitin- and proteasome-binding factors Rad23 and valosin-containing protein (VCP/p97), findings that support the hypothesis that ataxin-3 is a proteasome-associated factor that mediates the degradation of ubiquitinated proteins.

Down-regulation of genes in the lysosomal and ubiquitin-proteasome proteolytic pathways in calpain-3-deficient muscle

Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NFκB/IκB pathway. As this phenotype is mainly atrophic, this study was performed to determine whether protein turnover and/or proteolytic gene expression was altered in muscles following calpain-3 deficiency. In vitro rates of protein turnover and of substrate ubiquitination, cathepsin B and B+L activities, and mRNA levels for several proteolytic genes were measured in skeletal muscles from 4–5 month-old control and calpain-3 knockout mice. Rates of protein synthesis and breakdown, cathepsin activities, and rates of substrate ubiquitination remained stable in muscles from calpain-3 deficient mice. However, and surprisingly, mRNA levels for cathepsin L, the 14-kDa ubiquitin-conjugating enzyme E2, and the C2 subunit of the 20S proteasome decreased by ∼47% ( P<0.005) in the gastrocnemius muscle from calpain-3 deficient mice. In contrast, muscle mRNA levels for ubiquitin and subunit S5a of the 26S proteasome were unaffected by calpain-3 deficiency. Taken together these data demonstrate that the expression of some genes that are involved in distinct proteolytic pathways is selectively and coordinately down-regulated without any effect on proteolysis. This suggests new pathophysiological hypotheses, e.g. a lack of maturation of NFκB precursor and/or a defect in specific substrate targeting.

Early Holocene brackish and marine facies in the Fehmarn Belt, southwest Baltic Sea: depositional processes revealed by high-resolution seismic and core analysis

Holocene stratigraphy is established from 3.5-kHz seismic reflection data and a number of vibro cores across the narrow Fehmarn Belt between Germany and Denmark. Using detailed line drawings, texture, seismic and lithofacies analyses in a selected part of the study area, a distinct depositional unit 5 was identified. The unit was subdivided into sub-units 5a to 5j due to a different seismic texture and facies pattern. The base of the unit 5 is generally smooth but with step-like parts which are interpreted to result from tectonism. In each sub-unit the seismic facies association includes mound, oblique seismic facies and within channel deposits sigmoid, shingled and parallel facies. The sub-unit represents offset stacked lagoonal and spit deposits deposited under conditions of progressive sea-level rise. Sub-units 5a to 5h show a change in depositional style from fluvial domination to gradually more wave-influenced. Thus, the dynamic evolution of the shallow marine sub-sea-floor environment and changes in depositional conditions are revealed by high-resolution seismic facies associations.

Ubiquitin: no longer the chosen one

Since the discovery that proteins can be modified by the covalent attachment of ubiquitin, other ubiquitin-like proteins (ULPs) have been described. ULPs can also be used for posttranslational modifications of substrate proteins in a process similar to ubiquitination. However, ubiquitin was considered unique as only ubiquitin is able to form chains comprising multiple moieties, and polyubiquitin chains linked by Lys48 target proteins for degradation by the 26S proteasome. Mono-ubiquitination, as well as modification by the other ULPs, was considered to serve other, non-proteolytic, roles. Now, this simplistic point of view has been challenged by Yeh and colleagues [1xTargeting of NEDD8 and its conjugates for proteasomal degradation by NUB1. Kamitani, T. et al. J. Biol. Chem. 2001; 276: 46655–46660Crossref | PubMed | Scopus (93)See all References[1].Covalent modification by a ULP called NEDD8 (i.e. neddylation) was shown to target a specific set of proteins for degradation by the 26S proteasome. Neddylated proteins bind to an adaptor protein called NUB1. NUB1 possesses a proteasome-interacting motif known as a ubiquitin-like domain and therefore specifically binds to the ubiquitin-binding component of the 26S proteasome (the S5a subunit of the 19S cap). Neddylated proteins have been found associated with the 26S proteasome in vivo. NUB1 downregulates the expression of NEDD8 and its conjugates, but this effect can be prevented by the use of proteasome inhibitors. Altogether, these data suggest that neddylated proteins are targeted to the 26S proteasome for destruction.Although many proteins are neddylated, the only known ones are cullins. NEDD8 itself – unlike ubiquitin – appears to be degraded by the proteasomes.These findings are important because they show that ubiquitination is no longer the only method of protein tagging for proteasome-dependent destruction. Moreover, through neddylation of the cullins, various crucial pathways might be controlled. For example, neddylation of cullin1, a component of the ubiquitin-ligase SCF complex, could regulate the NF-κB signaling pathway and the cell cycle by indirectly controlling the ubiquitination and subsequent degradation of IκBα, cyclins and cyclin-dependent kinase inhibitors.

Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a.

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.

Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin–proteasome-dependent proteolysis in cancer and septic rats

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-α (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19S regulatory complex of the 26S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub—proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.