The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.

Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, enriched in peptides of mitochondrial origin, appears independent of the UPS. A further ~10% of peptides were found to be dependent on the proteasome but independent of ubiquitination for their generation. Notably, clinically achievable partial inhibition of the proteasome resulted in display of atypical peptides. Our results suggest that generation of MHC class I•peptide complexes is more complex than previously recognized, with UPS-dependent and UPS-independent components; paradoxically, alternative protein degradation pathways also generate class I peptides when canonical pathways are impaired.

Comparison between all-on-four and all-on-six treatment concepts on stress distribution for full-mouth rehabilitation using three-dimensional finite element analysis: A biomechanical study.

Purpose: The current study intended to provide a comparison of biomechanical behaviors of two different treatment concepts for full-mouth rehabilitation with dental implants placed according to the "All-on-four" concept and "All-on-six" concept with analysis of the stress patterns of the implant support system using three-dimensional finite element analysis (FEA). Materials and Methods: The edentulous mandible was treated with two different implant designs. "All-on-Four" implant placement concept was used in Model 1 with two central axial implants and two distally tilted implants at 17° and in Model 2, "All-on-Six" concept was applied with six vertically placed implants. Individual vertical and horizontal load of 100 N and oblique load of 141 N at 45° was applied to all implants. To evaluate and compare the results in terms of maximum principal stress, we used FEA. Results: All-on-six showed smaller maximum principal stress values on the cortical bone and implants. However, maximum principal stress values obtained on trabecular bone was smaller in the All-on-four design for vertical and horizontal loading conditions. Conclusions: The All-on-six approach showed more favorable biomechanical behavior.

Exploiting Ubiquitin Ligases for Induced Target Degradation as an Antiviral Strategy.

Posttranslational modifications of targeted substrates alter their cellular fate. Ubiquitin is a highly conserved and ubiquitous covalent modifier protein that tags substrates with a single molecule or with a polyubiquitin chain. Monoubiquitination affects trafficking and signaling patterns of modified proteins. In contrast, polyubiquitination, particularly K48-linked polyubiquitination, targets the protein for degradation by the Ubiquitin-Proteasome System (UPS) resulting in a committed fate through irreversible inactivation of substrate. Given the diversity of cellular functions impacted by ubiquitination, it is no surprise that the wily pathogenic viruses have co-opted the UPS in myriad ways to ensure their survival. In this review, I describe viral exploitation of nondegradative ubiquitin signaling pathways to effect entry, replication, and egress. Additionally, viruses also harness the UPS to degrade antiviral cellular host factors. Finally, I describe how we can exploit the same proteolytic machinery to enable PROTACs (Proteolysis-Targeting Chimeras) to degrade essential viral proteins. Successful implementation of this modality will add to the arsenal of emerging antiviral therapies.

Harnessing the Power of Proteolysis for Targeted Protein Inactivation.

Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients.

Evaluation and comparison of stress distribution around periodontally compromised mobile teeth splinted with different materials: Three-dimensional finite element analysis.

BACKGROUND: Progressive attachment loss around the teeth because of periodontal disease can result in increased tooth mobility. This adversely affects patient's comfort, function, and esthetics. Periodontal splinting helps in accomplishing stability by redistributing the functional and parafunctional forces. There are various materials that have been used for periodontal splinting. Fiber-reinforced composite, composite resin, and metal-reinforced composite are often used as splinting materials for periodontally compromised teeth. In our study, a comparison was done among these materials for their ability to distribute the stresses at different bone levels in mobile lower incisors splinted together with canines. MATERIALS AND METHODS: Five patients of age group 25-50 years with Grade 2 and 3 mobile incisors having 40% or more bone loss and firm canines with optimal bone support were selected. From the computed tomography scan of each patient, three models were developed demonstrating splinting of mandibular incisors and canines with metal-reinforced composite, fiber-reinforced composite, and composite resin. So in total, 15 models were developed and each one of them was subjected to vertical and transverse loads of 150 N. Pattern of stress distribution was observed in these models using three-dimensional finite element analysis. RESULTS: After splinting, the stress on the canine increased when bone levels around incisors decreased while stress on incisors reduced. CONCLUSION: Tested splinting materials were successful in stress distribution, and metal-reinforced composite was found to be better than the other splinting materials.

Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes.

Ribosomal surveillance pathways scan for ribosomes that are transiently paused or terminally stalled owing to structural elements in mRNAs or nascent chain sequences1, 2. Some stalls in budding yeast are sensed by the GTPase Hbs1, which loads Dom34, a catalytically inactive member of the archaeo-eukaryotic release factor 1 superfamily. Hbs1-Dom34 and the ATPase Rli1 dissociate stalled ribosomes into 40S and 60S subunits. However, the 60S subunits retain the peptidyl-tRNA nascent chains, which recruit the ribosome quality control complex that consists of Rqc1-Rqc2-Ltn1-Cdc48-Ufd1-Npl4. Nascent chains ubiquitylated by the E3 ubiquitin ligase Ltn1 are extracted from the 60S subunit by the ATPase Cdc48-Ufd1-Npl4 and presented to the 26S proteasome for degradation3-9. Failure to degrade the nascent chains leads to protein aggregation and proteotoxic stress in yeast and neurodegeneration in mice10-14. Despite intensive investigations on the ribosome quality control pathway, it is not known how the tRNA is hydrolysed from the ubiquitylated nascent chain before its degradation. Here we show that the Cdc48 adaptor Vms1 is a peptidyl-tRNA hydrolase. Similar to classical eukaryotic release factor 1, Vms1 activity is dependent on a conserved catalytic glutamine. Evolutionary analysis indicates that yeast Vms1 is the founding member of a clade of eukaryotic release factor 1 homologues that we designate the Vms1-like release factor 1 clade.

Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome.

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.DOI:http://dx.doi.org/10.7554/eLife.00308.001.

NEDD8 links cullin-RING ubiquitin ligase function to the p97 pathway.

The AAA+ ATPase p97 and its UBA-UBX cofactors are thought to extract ubiquitinated proteins from membranes or protein complexes as a prelude to their degradation. However, for many cofactors ubiquitinated targets have not yet been identified, leaving their biological function unclear. Previous analysis has linked the p97 pathway to cullin-RING ubiquitin ligases (CRLs); here we demonstrate that the human p97 cofactor UBXD7 mediates the p97-CRL interaction through its conserved ubiquitin-interacting motif (UIM). UBXD7 and its yeast ortholog, Ubx5, associate only with the active, NEDD8- or Rub1-modified form of cullins. Disruption of the Ubx5 UIM results in a loss of CRL binding and consequently impedes degradation of a Cul3 substrate. These results uncover an unexpected and conserved role for NEDD8 in linking CRL ubiquitin ligase function to the p97 pathway.

The TFIIH subunit Tfb3 regulates cullin neddylation.

Cullin proteins are scaffolds for the assembly of multisubunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING domain of Hrt1 nor required the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 and the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING domain protein Tfb3 controls activation of a subset of cullins.

Cdc48/p97 mediates UV-dependent turnover of RNA Pol II.

Cdc48/p97 is an essential ATPase whose role in targeting substrates to the ubiquitin-proteasome system (UPS) remains unclear. Existing models posit that Cdc48 acts upstream of UPS receptors. To address this hypothesis, we examined the association of ubiquitin (Ub) conjugates with 26S proteasomes. Unexpectedly, proteasomes isolated from cdc48 mutants contain high levels of Ub conjugates, and mass spectrometry identified numerous nonproteasomal proteins, including Rpb1, the largest subunit of RNA Pol II. UV-induced turnover of Rpb1 depends upon Cdc48-Ufd1-Npl4, Ubx4, and the uncharacterized adaptor Ubx5. Ubiquitinated Rpb1, proteasomes, and Cdc48 accumulate on chromatin in UV-treated wild-type cells, and the former two accumulate to higher levels in mutant cells, suggesting that degradation of Rpb1 is facilitated by Cdc48 at sites of stalled transcription. These data reveal an intimate coupling of function between proteasomes and Cdc48 that we suggest is necessary to sustain processive degradation of unstable subunits of some macromolecular protein complexes.

Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

Components of the ubiquitin-proteasome pathway compete for surfaces on Rad23 family proteins.

BACKGROUND: The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain. RESULTS: In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome. CONCLUSION: We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting.

Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins.

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal beta-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.

Assaying degradation and deubiquitination of a ubiquitinated substrate by purified 26S proteasomes.

The 26S proteasome is a multisubunit complex that catalyzes ATP-dependent proteolysis of cellular proteins. It eliminates misfolded proteins, as well as labile regulatory proteins, thereby serving a central role in maintaining cellular homeostasis. The bulk of the known substrates of the 26S proteasome are earmarked for proteolysis by covalent modification with a multiubiquitin chain, which is recognized by specific receptors. Once targeted, the substrate is deubiquitinated and degraded by the 26S proteasome. This chapter describes assays that monitor ATP- and ubiquitin-dependent proteolysis of the S-Cdk inhibitor Sic1.

Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.

Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.

Recruitment of ubiquitinated proteins to the 26S proteasome lies at the heart of the ubiquitin-proteasome system (UPS). Genetic studies suggest a role for the multiubiquitin chain binding proteins (MCBPs) Rad23 and Rpn10 in recruitment, but biochemical studies implicate the Rpt5 ATPase. We addressed this issue by analyzing degradation of the ubiquitinated Cdk inhibitor Sic1 (UbSic1) in vitro. Mutant rpn10Delta and rad23Delta proteasomes failed to bind or degrade UbSic1. Although Rpn10 or Rad23 restored UbSic1 recruitment to either mutant, rescue of degradation by Rad23 uncovered a requirement for the VWA domain of Rpn10. In vivo analyses confirmed that Rad23 and the multiubiquitin binding domain of Rpn10 contribute to Sic1 degradation. Turnover studies of multiple UPS substrates uncovered an unexpected degree of specificity in their requirements for MCBPs. We propose that recruitment of substrates to the proteasome by MCBPs provides an additional layer of substrate selectivity in the UPS.

Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation.

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCF(beta-TRCP)-binding phosphopeptide derived from IkappaBalpha linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the alpha isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.

Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome.

The 26S proteasome mediates degradation of ubiquitin-conjugated proteins. Although ubiquitin is recycled from proteasome substrates, the molecular basis of deubiquitination at the proteasome and its relation to substrate degradation remain unknown. The Rpn11 subunit of the proteasome lid subcomplex contains a highly conserved Jab1/MPN domain-associated metalloisopeptidase (JAMM) motif-EX(n)HXHX(10)D. Mutation of the predicted active-site histidines to alanine (rpn11AXA) was lethal and stabilized ubiquitin pathway substrates in yeast. Rpn11(AXA) mutant proteasomes assembled normally but failed to either deubiquitinate or degrade ubiquitinated Sic1 in vitro. Our findings reveal an unexpected coupling between substrate deubiquitination and degradation and suggest a unifying rationale for the presence of the lid in eukaryotic proteasomes.