James A. Nathan

Muscle wasting in disease: molecular mechanisms and promising therapies

Atrophy occurs in specific muscles with inactivity (for example, during plaster cast immobilization) or denervation (for example, in patients with spinal cord injuries). Muscle wasting occurs systemically in older people (a condition known as sarcopenia); as a physiological response to fasting or malnutrition; and in many diseases, including chronic obstructive pulmonary disorder, cancer-associated cachexia, diabetes, renal failure, cardiac failure, Cushing syndrome, sepsis, burns and trauma. The rapid loss of muscle mass and strength primarily results from excessive protein breakdown, which is often accompanied by reduced protein synthesis. This loss of muscle function can lead to reduced quality of life, increased morbidity and mortality. Exercise is the only accepted approach to prevent or slow atrophy. However, several promising therapeutic agents are in development, and major advances in our understanding of the cellular mechanisms that regulate the protein balance in muscle include the identification of several cytokines, particularly myostatin, and a common transcriptional programme that promotes muscle wasting. Here, we discuss these new insights and the rationally designed therapies that are emerging to combat muscle wasting.

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Although cellular proteins conjugated to K48‐linked Ub chains are targeted to proteasomes, proteins conjugated to K63‐ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48‐ and K63‐ubiquitinated substrates similarly. Therefore, we investigated why K63‐ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63‐ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48‐ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48‐ or K63‐ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal‐lysosomal pathway.

The trafficking and regulation of membrane receptors by the RING-CH ubiquitin E3 ligases.

Ubiquitylation of membrane receptors is recognised as a critical post-translational modification, governing their regulation and function. Following ubiquitylation, membrane proteins may be internalised, recycled or degraded via lysosomal or proteasomal pathways. Viruses have appropriated these cellular pathways as a mechanism of immune evasion. RING (really interesting new gene)-CH ubiquitin E3 ligases were initially identified from the Kaposis associated herpesvirus (KSHV) and their founding members, K3 and K5, downregulate several critical immunoreceptors to prevent detection by the host immune system. K3 promotes formation of lysine-63 linked polyubiquitin chains on MHC Class I, signalling Class I internalisation and endolysosomal degradation. K5 targets multiple immunoreceptors, including MHC Class I, CD86, intracellular adhesion molecule (ICAM) 1 and MHC Class I-related chain (MIC)-A/B, thereby preventing detection from cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cellular homologues of K3 and K5, the Membrane Associated RING-CH (MARCH) genes, represent eleven proteins that also appear to be important in the downregulation of membrane receptors. While overexpression of several MARCH genes downregulate cell surface receptors such as MHC Class I, MHC Class II, CD86 and ICAM 1, determining their physiological roles has proved difficult. Elucidating the transcriptional regulation, localisation and trafficking of MARCH genes may provide insights into their cellular functions.

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry

Prion diseases are associated with the conversion of cellular prion protein (PrPC) to toxic β‐sheet isoforms (PrPSc), which are reported to inhibit the ubiquitin‐proteasome system (UPS). Accordingly, UPS substrates accumulate in prion‐infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. β‐PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated β‐PrP reduced the 20S proteasomes basal peptidase activity, and the enhanced activity induced by C‐terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α‐ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded β‐sheet‐rich proteins accumulate.

The Ubiquitin E3 Ligase MARCH7 is Differentially Regulated by the Deubiquitylating Enzymes USP7 and USP9X

Protein modification by one or more ubiquitin chains serves a critical signalling function across a wide range of cellular processes. Specificity within this system is conferred by ubiquitin E3 ligases, which target the substrates. Their activity is balanced by deubiquitylating enzymes (DUBs), which remove ubiquitin from both substrates and ligases. The RING‐CH ligases were initially identified as viral immunoevasins involved in the downregulation of immunoreceptors. Their cellular orthologues, the Membrane‐Associated RING‐CH (MARCH) family represent a subgroup of the classical RING genes. Unlike their viral counterparts, the cellular RING‐CH proteins appear highly regulated, and one of these in particular, MARCH7, was of interest because of a potential role in neuronal development and lymphocyte proliferation. Difficulties in detection and expression of this orphan ligase lead us to search for cellular cofactors involved in MARCH7 stability. In this study, we show that MARCH7 readily undergoes autoubiquitylation and associates with two deubiquitylating enzymes – ubiquitin‐specific protease (USP)9X in the cytosol and USP7 in the nucleus. Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively. We therefore demonstrate compartment‐specific regulation of this E3 ligase through recruitment of site‐specific DUBs.

The ATP Costs and Time Required to Degrade Ubiquitinated Proteins by the 26 S Proteasome

Background: Multiple steps in the degradation of ubiquitinated proteins by the 26 S proteasome require ATP. Results: The six ATPase subunits of the proteasome function in a cyclic manner. Rates of degradation of ubiquitinated proteins are directly proportional to rates of ATP hydrolysis. Conclusion: A specific number of ATPs are consumed in degrading a ubiquitinated substrate. Significance: Polypeptide structure determines the time required and ATP consumed in degrading ubiquitin conjugates. The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2–3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50–80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost.

Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins

Summary Immunoproteasomes are alternative forms of proteasomes that have an enhanced ability to generate antigenic peptides. Recently, Seifert and colleagues reported surprising observations concerning the functions of immunoproteasomes and cellular responses to interferon-γ: (1) that immunoproteasomes degrade ubiquitinated proteins faster than the constitutive proteasomes, (2) that polyubiquitin conjugates accumulate after interferon-γ treatment but then are preferentially degraded by immunoproteasomes, and (3) that immunoproteasome deficiency causes the formation of inclusions and more severe experimental autoimmune encephalomyelitis (EAE). In contrast, we find that polyubiquitin conjugates do not transiently accumulate following IFNγ-treatment and that immunoproteasomes do not prevent the formation of intracellular inclusions or protect against EAE. Furthermore, purified 26S constitutive and immunoproteasomes bind ubiquitin conjugates similarly and degrade them at similar rates. We conclude that, although immunoproteasomes can increase the generation of peptides appropriate for MHC class I presentation, they do not degrade ubiquitinated proteins more efficiently than constitutive particles.

The recognition of ubiquitinated proteins by the proteasome.

The ability of ubiquitin to form up to eight different polyubiquitin chain linkages generates complexity within the ubiquitin proteasome system, and accounts for the diverse roles of ubiquitination within the cell. Understanding how each type of ubiquitin linkage is correctly interpreted by ubiquitin binding proteins provides important insights into the link between chain recognition and cellular fate. A major function of ubiquitination is to signal degradation of intracellular proteins by the 26S proteasome. Lysine-48 (K48) linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation, but recent studies show a role for other ubiquitin linked chains in facilitating degradation by the 26S proteasome. Here, we review how different types of polyubiquitin linkage bind to ubiquitin receptors on the 26S proteasome, how they signal degradation and discuss the implications of ubiquitin chain linkage in regulating protein breakdown by the proteasome.

TMEM129 is a Derlin-1 associated ERAD E3 ligase essential for virus-induced degradation of MHC-I

Significance Newly synthesized proteins undergo a strict quality-control checkpoint, and misfolded secretory proteins are targeted across the endoplasmic reticulum membrane back to the cytosol for proteasome degradation. This process requires tagging errant proteins with ubiquitin by an E3 ubiquitin ligase. In a genetic screen we identified TMEM129 as a novel and unusual E3 ligase. TMEM129 is hijacked by the human cytomegalovirus to degrade MHC-I signaling molecules and avert immune recognition of the infected cell. We suggest TMEM129 is an important ligase in the turnover of misfolded secretory proteins within a novel endoplasmic reticulum-associated degradation complex. The US11 gene product of human cytomegalovirus promotes viral immune evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US11 initiates dislocation of newly translocated MHC I from the ER to the cytosol for proteasome-mediated degradation. Despite the critical role for ubiquitin in this degradation pathway, the responsible E3 ligase is unknown. In a forward genetic screen for host ERAD components hijacked by US11 in near-haploid KBM7 cells, we identified TMEM129, an uncharacterized polytopic membrane protein. TMEM129 is essential and rate-limiting for US11-mediated MHC-I degradation and acts as a novel ER resident E3 ubiquitin ligase. TMEM129 contains an unusual cysteine-only RING with intrinsic E3 ligase activity and is recruited to US11 via Derlin-1. Together with its E2 conjugase Ube2J2, TMEM129 is responsible for the ubiquitination, dislocation, and subsequent degradation of US11-associated MHC-I. US11 engages two degradation pathways: a Derlin-1/TMEM129–dependent pathway required for MHC-I degradation and a SEL1L/HRD1-dependent pathway required for “free” US11 degradation. Our data show that TMEM129 is a novel ERAD E3 ligase and the central component of a novel mammalian ERAD complex.

Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

Intramembrane proteolytic cleavage by signal peptide peptidase is required for the turnover of some ER-resident, tail-anchored membrane proteins.