Structural insights into specificity and diversity in mechanisms of ubiquitin recognition by ubiquitin-binding domains

UBDs [Ub (ubiquitin)-binding domains], which are typically small protein motifs of <50 residues, are used by receptor proteins to transduce post-translational Ub modifications in a wide range of biological processes, including NF-κB (nuclear factor κB) signalling and proteasomal degradation pathways. More than 20 families of UBDs have now been characterized in structural detail and, although many recognize the canonical Ile44/Val70-binding patch on Ub, a smaller number have alternative Ub-recognition sites. The A20 Znf (A20-like zinc finger) of the ZNF216 protein is one of the latter and binds with high affinity to a polar site on Ub centred around Asp58/Gln62. ZNF216 shares some biological function with p62, with both linked to NF-κB signal activation and as shuttle proteins in proteasomal degradation pathways. The UBA domain (Ub-associated domain) of p62, although binding to Ub through the Ile44/Val70 patch, is unique in forming a stable dimer that negatively regulates Ub recognition. We show that the A20 Znf and UBA domain are able to form a ternary complex through independent interactions with a single Ub molecule, supporting functional models for Ub as a 'hub' for mediating multi-protein complex assembly and for enhancing signalling specificity.     

Haploinsuffciency for Znf9 in Znf9+/- mice is associated with multiorgan abnormalities resembling myotonic dystrophy

Myotonic dystrophy type 2 is caused by a (CCTG)/(CCUG)n repeat expansion in the first intron of the ZNF9 gene. The pathomechanism for the myotonic dystrophies is not well understood and the role of ZNF9 in myotonic dystrophy type 2 pathogenesis has not been fully clarified. We characterized Znf9+/- mice, in which the expression of Znf9 was significantly decreased, and found that their phenotype reflects many of the features of myotonic dystrophy, including muscle histological morphology, and myotonic discharges and heart conduction abnormalities, shown by electromyography and electrocardiogram analysis, respectively. Znf9 is normally highly expressed in heart and skeletal muscle, where skeletal muscle chloride channel 1 (Clc1) plays an important role. Clc1 expression was dramatically decreased in Znf9+/- mice. Znf9 transgenic mice raised Znf9 and Clc1 expression and rescued the myotonic dystrophy phenotype in Znf9+/- mice. Our results suggest that the Znf9 haploinsufficiency contributes to the myotonic dystrophy phenotype in Znf9+/- mice.     

Independent interactions of ubiquitin-binding domains in a ubiquitin-mediated ternary complex

Ubiquitin (Ub) modifications are transduced by receptor proteins that use Ub-binding domains (UBDs) to recognize distinct interaction faces on the Ub surface. We report the nuclear magnetic resonance (NMR) solution structures of the A20-like zinc finger (A20 Znf) UBD of the Ub receptor ZNF216, and its complex with Ub, and show that the binding surface on Ub centered on Asp58 leaves the canonical hydrophobic Ile44 patch free to participate in additional interactions. We have modeled ternary complexes of the different families of UBDs and show that while many are expected to bind competitively to the same Ile44 surface or show steric incompatibility, other combinations (in particular, those involving the A20 Znf domain) are consistent with a single Ub moiety simultaneously participating in multiple interactions with different UBDs. We subsequently demonstrate by NMR that the A20 Znf domain of ZNF216 and the UBA domain of the p62 protein (an Ile44-binding UBD), which function in the same biological pathways, are able to form such a Ub-mediated ternary complex through independent interactions with a single Ub. This work supports an emerging concept of Ub acting as a scaffold to mediate multiprotein complex assembly.     

A RANKL-inducible gene Znf216 in osteoclast differentiation

Osteoclasts possess catabolic activity in mineralized tissues and are involved in bone remodeling coordinating with osteoblasts. Although the pathway using receptor and activator of NF-kappa B (RANK) and its ligand, RANKL, is known to be essential for osteoclast differentiation, their precise mechanisms are not fully understood. Using DNA microarray technology, we searched for genes that were up-regulated after RANKL stimulation in the macrophage cell line, RAW264.7 cells. A gene, Znf216, which encodes a zinc-finger protein, was detected among those genes up-regulated after RANKL stimulation. Expression of Znf216 was also induced by other cytokines such as TNFalpha and IL-1beta. Although ectopic expression of full-length ZNF216 abrogated osteoclast differentiation, its truncated forms accelerated it. No significant inhibitory effect on the NF-kappa B pathway was observed, however. These results suggest that ZNF216 is a potent inhibitory factor for osteoclast differentiation and that the mechanism is unlikely due to direct attenuation of the NF-kappa B pathway.     

A RANKL-Inducible Gene Znf216 in Osteoclast Differentiation

Osteoclasts possess catabolic activity in mineralized tissues and are involved in bone remodeling coordinating with osteoblasts. Although the pathway using receptor and activator of NF-κ B (RANK) and its ligand, RANKL, is known to be essential for osteoclast differentiation, their precise mechanisms are not fully understood. Using DNA microarray technology, we searched for genes that were up-regulated after RANKL stimulation in the macrophage cell line, RAW264.7 cells. A gene, Znf216, which encodes a zinc-finger protein, was detected among those genes up-regulated after RANKL stimulation. Expression of Znf216 was also induced by other cytokines such as TNFα and IL-1β. Although ectopic expression of full-length ZNF216 abrogated osteoclast differentiation, its truncated forms accelerated it. No significant inhibitory effect on the NF-κ B pathway was observed, however. These results suggest that ZNF216 is a potent inhibitory factor for osteoclast differentiation and that the mechanism is unlikely due to direct attenuation of the NF-κ B pathway.     

NF-κB activation and polyubiquitin conjugation are required for pulmonary inflammation-induced diaphragm atrophy

Loss of diaphragm muscle strength in inflammatory lung disease contributes to mortality and is associated with diaphragm fiber atrophy. Ubiquitin (Ub) 26S-proteasome system (UPS)-dependent protein breakdown, which mediates muscle atrophy in a number of physiological and pathological conditions, is elevated in diaphragm muscle of patients with chronic obstructive pulmonary disease. Nuclear factor kappa B (NF-κB), an essential regulator of many inflammatory processes, has been implicated in the regulation of poly-Ub conjugation of muscle proteins targeted for proteolysis by the UPS. Here, we test if NF-κB activation in diaphragm muscle and subsequent protein degradation by the UPS are required for pulmonary inflammation-induced diaphragm atrophy. Acute pulmonary inflammation was induced in mice by intratracheal lipopolysaccharide instillation. Fiber cross-sectional area, ex vivo tyrosine release, protein poly-Ub conjugation, and inflammatory signaling were determined in diaphragm muscle. The contribution of NF-κB or the UPS to diaphragm atrophy was assessed in mice with intact or genetically repressed NF-κB signaling or attenuated poly-Ub conjugation, respectively. Acute pulmonary inflammation resulted in diaphragm atrophy measured by reduced muscle fiber cross-sectional area. This was accompanied by diaphragm NF-κB activation, and proteolysis, measured by tyrosine release from the diaphragm. Poly-Ub conjugation was increased in diaphragm, as was the expression of muscle-specific E3 Ub ligases. Genetic suppression of poly-Ub conjugation prevented inflammation-induced diaphragm muscle atrophy, as did muscle-specific inhibition of NF-κB signaling. In conclusion, the present study is the first to demonstrate that diaphragm muscle atrophy, resulting from acute pulmonary inflammation, requires NF-κB activation and UPS-mediated protein degradation.     

Molecular determinants of skeletal muscle mass: getting the "AKT" together

Skeletal muscle is the most abundant tissue in the human body and its normal physiology plays a fundamental role in health and disease. During many disease states, a dramatic loss of skeletal muscle mass (atrophy) is observed. In contrast, physical exercise is capable of producing significant increases in muscle mass (hypertrophy). Maintenance of skeletal muscle mass is often viewed as the net result of the balance between two separate processes, namely protein synthesis and protein degradation. However, these two biochemical processes are not occurring independent of each other but they rather appear to be finely coordinated by a web of intricate signaling networks. Such signaling networks are in charge of executing environmental and cellular cues that will ultimate determine whether muscle proteins are synthesized or degraded. In this review, recent findings are discussed demonstrating that the AKT1/FOXOs/Atrogin-1(MAFbx)/MuRF1 signaling network plays an important role in the progression of skeletal muscle atrophy. These novel findings highlight an important mechanism that coordinates the activation of the protein synthesis machinery with the activation of a genetic program responsible for the degradation of muscle proteins during skeletal muscle atrophy.     

Proteasome function is required to maintain muscle cellular architecture

BACKGROUND INFORMATION: Protein degradation via the UPS (ubiquitin-proteasome system) plays critical roles in muscle metabolism and signalling pathways. The present study investigates temporal requirements of the UPS in muscle using conditional expression of mutant proteasome beta subunits to cause targeted inhibition of proteasome function. RESULTS AND CONCLUSIONS: The Drosophila GeneSwitch system was used, with analyses of the well-characterized larval somatic body wall muscles. This method acutely disrupts proteasome function and causes rapid accumulation of polyubiquitinated proteins, specifically within the muscle. Within 12 h of transgenic proteasome inhibition, there was a gross disorganization of muscle architecture and prominent muscle atrophy, progressing to the arrest of all co-ordinated movement by 24 h. Progressive muscle architecture changes include rapid loss of sarcomere organization, loss of nuclei spacing/patterning, vacuole formation and the accumulation of nuclear and cytoplasmic aggregates at the ultrastructural level. At the neuromuscular junction, the highly specialized muscle membrane folds of the subsynaptic reticulum were rapidly lost. Within 24 h after transgenic proteasome inhibition, muscles contained numerous autophagosomes and displayed highly elevated expression of the endoplasmic reticulum chaperone GRP78 (glucose-regulated protein of 78 kDa), indicating that the loss of muscle maintenance correlates with induction of the unfolded protein response. Taken together, these results demonstrate that the UPS is acutely required for maintenance of muscle and neuromuscular junction architecture, and provides a Drosophila genetic model to mechanistically evaluate this requirement.     

自噬和泛素-蛋白酶体系统之间的交联

自噬和泛素-蛋白酶体系统作为细胞内最重要的两大降解途径,对细胞稳态及细胞正常生理功能的维持都具有十分重要的作用。目前,越来越多的证据显示,这两大降解途径之间存在多种交联方式。首先,自噬和泛素-蛋白酶体系统都能以泛素作为共同标签,从而将泛素化底物降解;其次,泛素化的蛋白酶体可以通过自噬被清除,自噬相关蛋白质也可以通过蛋白酶体系统被降解;再次,这两条途径在细胞内能协同降解同一种底物;最后,它们之间可以相互调节活性,任一条途径被干扰都将影响另一条途径的活性。自噬和泛素-蛋白酶体系统之间的交联对细胞稳态的维持至关重要。交联失调不仅导致细胞功能异常,还可引起多种疾病的发生。本文主要对自噬和泛素-蛋白酶体系统之间的交联方式及其分子机制进行阐述,有助于深入了解细胞的分解代谢过程,进一步理解细胞稳态的维持机制,继而加深对相关疾病病理机制的认识。     

Unmethylated state of 5′ upstream CpG islands may be necessary but not sufficient for the testis-enriched expression of ZNF230/Znf230

The testis-enriched genes ZNF230/Znf230 are located on human chromosome 11p15/mouse chromosome 7 near conserved imprinting control regions. Typical CpG islands (CGIs) extend from the promoter to the first exon in each of these genes. To investigate the correlation between the methylation status of the above CGIs and the expression patterns of the two genes, we performed bisulfite genomic sequencing of genomic DNA from human and mouse tissues and cells. The results showed that the CGIs of ZNF230/Znf230 were completely unmethylated in all selected tissues and cells, regardless of the expression levels of the two genes. Further experiments using Znf230-second-exon-knockout mice to investigate the imprinting status of Znf230 showed that its expression was not affected by genomic imprinting. However, an in vitro methylation assay illustrated that the methylation of these CpG sites could repress the expression of the luciferase reporter gene. Furthermore, chromatin immunoprecipitation with anti-Specificity protein 1 (Sp1) antibody showed that Sp1 could bind to the CGIs in the ZNF230/Znf230 gene promoter. Thus, we propose that the unmethylated state of ZNF230/Znf230 CGIs may be a prerequisite for their expression but not sufficient for their abundant expression in the testis, and that Sp1 binding may be one factor involved in preserving the methylation-free state of ZNF230/Znf230 CGIs.     

The dependency of autophagy and ubiquitin proteasome system during skeletal muscle atrophy

Among the four proteolytic systems in the cell, autophagy and the ubiquitin-proteasome system (UPS) are the main proteolytic events that allow for the removal of cell debris and proteins to maintain cellular homeostasis. Previous studies have revealed that these systems perform their functions independently of each other. However, recent studies indicate the existence of regulatory interactions between these proteolytic systems via ubiquitinated tags and a reciprocal regulation mechanism with several crosstalk points. UPS plays an important role in the elimination of short-lived/soluble misfolded proteins, whereas autophagy eliminates defective organelles and persistent insoluble protein aggregates. Both of these systems seem to act independently; however, disruption of one pathway affects the activity of the other pathway and contributes to different pathological conditions. This review summarizes the recent findings on direct and indirect dependencies of autophagy and UPS and their execution at the molecular level along with the important drug targets in skeletal muscle atrophy.     

Metabolism of skeletal muscle proteins in experimental hypokinesia in rats

Using a radioindicator method, the metabolism of sarcoplasmic and myofibrillar proteins was studied in vivo at different stages of hypokinesia in rats. It was shown that the true muscle atrophy and the lowered content of both protein fractions during the first two weeks are due to sharp inhibition of sarcoplasmic protein biosynthesis as well as to deceleration of biosynthesis and acceleration of degradation of actomyosin. In hypokinesia the muscle mass does not increase within 3-8 weeks largely due to the acceleration of degradation of the de novo synthesized components of contractile proteins. In early hypokinesia a stressory mechanism plays an essential role in the pathogenesis of protein metabolism disturbances.     

Insights into the molecular composition of endogenous unanchored polyubiquitin chains

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.     

Protein homeostasis and aging: role of ubiquitin protein ligases

Protein homeostasis is fundamental in normal cellular function and cell survival. The ubiquitin-proteasome system (UPS) plays a central role in maintaining the protein homeostasis network through selective elimination of misfolded and damaged proteins. Impaired function of UPS is implicated in normal aging process and also in several age-related neurodegenerative disorders that are characterized by increased accumulation oxidatively modified proteins and protein aggregates. Growing literature also indicate the potential role of various ubiquitin protein ligases in the regulation of aging process by enhancing the degradation of either central lifespan regulators or abnormally folded and damaged proteins. This review mainly focuses on our current understanding of the importance of UPS function in the regulation of normal aging process.     

Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala

Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.