Biallelic USP14 variants cause a syndromic neurodevelopmental disorder.

PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.

The role of SHMT2 in modulating lipid metabolism in hepatocytes via glycine-mediated mTOR activation.

Serine hydroxymethyltransferase 2 (SHMT2) converts serine into glycine in the mitochondrial matrix, transferring a methyl group to tetrahydrofolate. SHMT2 plays an important role in the maintenance of one-carbon metabolism. Previously, we found a negative correlation between the serine concentration and the progression of fatty liver disease (FLD). However, little is known about the role of SHMT2 in hepatic lipid metabolism. We established SHMT2 knockdown (KD) mouse primary hepatocytes using RNA interference to investigate the role of SHMT2 in lipid metabolism. SHMT2 KD hepatocytes showed decreased lipid accumulation with reduced glycine levels compared to the scramble cells, which was restored upon reintroducing SHMT2. SHMT2 KD hepatocytes showed downregulation of the mTOR/PPARÉ£ pathway with decreased gene expression related to lipogenesis and fatty acid uptake. Pharmacological activation of mTOR or PPARÉ£ overexpression blocked the inhibitory effect of SHMT2 KD on lipid accumulation. We also showed that glycine activated mTOR/PPARÉ£ signaling and identified glycine as a mediator of SHMT2-responsive lipid accumulation in hepatocytes. In conclusion, silencing SHMT2 in hepatocytes ameliorates lipid accumulation via the glycine-mediated mTOR/PPARÉ£ pathway. Our findings underscore the possibility of SHMT2 as a therapeutic target of FLD.

USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites.

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14's catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.

Intratracheal exposure to polyhexamethylene guanidine phosphate disrupts coordinate regulation of FXR-SHP-mediated cholesterol and bile acid homeostasis in mouse liver.

A public health crisis in the form of a significant incidence of fatal pulmonary disease caused by repeated use of humidifier disinfectants containing polyhexamethylene guanidine phosphate (PHMG) recently arose in Korea. Although the mechanisms of pulmonary fibrosis following respiratory exposure to PHMG are well described, distant-organ effect has not been reported. In this study, we investigated whether intratracheal administration of PHMG affects liver pathophysiology and metabolism. Our PHMG mouse model showed a significant decrease in liver cholesterol level. An mRNA-seq analysis of liver samples revealed an alteration in the gene expression associated with cholesterol biosynthesis and metabolism to bile acids. The expression of genes involved in cholesterol synthesis was decreased in a real-time PCR analysis. To our surprise, we found that the coordinate regulation of cholesterol and bile acid homeostasis was completely disrupted. Despite the decreased cholesterol synthesis and low bile acid levels, the farnesoid X receptor/small heterodimer partner pathway, which controls negative feedback of bile acid synthesis, was activated in PHMG mice. As a consequence, gene expression of Cyp7a1 and Cyp7b1, the rate-limiting enzymes of the classical and alternative pathways of bile acid synthesis, was significantly downregulated. Notably, the changes in gene expression were corroborated by the hepatic concentrations of the bile acids. These results suggest that respiratory exposure to PHMG could cause cholestatic liver injury by disrupting the physiological regulation of hepatic cholesterol and bile acid homeostasis.

Alignment changes after open-wedge high tibial osteotomy result in offloading in the patellofemoral joint: a SPECT/CT analysis.

PURPOSE: The patellofemoral (PF) joint may be adversely affected by medial open-wedge high tibial osteotomy (OWHTO). This study aimed to evaluate the PF compartmental changes using combined single-photon emission computed tomography (SPECT) and conventional computed tomography (CT) after OWHTO to provide clinical guidance regarding the PF joint pressure and force. METHODS: Patients with medial osteoarthritis and varus malalignment > 5° were treated using OWHTO. Patients with a minimum 2-year follow-up were included in the study. The patellar positions were evaluated based on the radiographic parameters. The changes in chondral lesions during second-look arthroscopic examination were evaluated, and the PF joint arthritis grade was recorded on patellar Merchant radiographs using Kellgren-Lawrence classification. The PF compartmental changes according to SPECT/CT analysis after OWHTO were evaluated in all patients. The scintigraphic uptake was graded on four scales. Patients were divided into improved and unimproved groups according to the PF compartmental grade using the SPECT/CT uptake grading system. RESULTS: At a mean follow-up period of 47.0 months (range 25-74 months), the mean mechanical femorotibial angle changed significantly from varus 6.3° (range 5-12°) to valgus 2.6° (range 0-8°); p < 0.001) postoperatively. The radiological parameters presenting patellar positions, including the tibial slope, patellar convergence angle, and lateral tilt angle, did not change significantly between the preoperative values and the 2-year follow-up values. The mean patellar height significantly decreased (0.07 ± 0.14, p = 0.001 according to the Blackburn-Peel index and 0.32 ± 0.23, p < 0.001 using the modified Insall-Salvati ratio). The average tibial tubercle to trochlear groove (TT-TG) distance significantly decreased from 14.1 to 12.2 mm (p < 0.001). The Q angle also significantly decreased from 9.8o to 7.7o (p = 0.008). Chondral lesions of the patella and trochlear groove revealed significant deterioration; at 2 years after OWHTO, the arthritic grades of the PF joints worsened significantly, as determined by radiography (p = 0.007). Scintigraphic uptake in the PF joint was significantly lower (from 2 to 1) at 2 years postoperatively compared to that immediately after the index operation (p < 0.001). Only 4 of 56 (7.1%) patients showed increased uptake. Comparison between the improved and unimproved groups according to scintigraphic uptake changes revealed that the changes in the cartilage status on the patellar undersurface and TT-TG distance were the most significant predictive factors of increased scintigraphic uptake in the PF joint after OWHTO. CONCLUSION: Alignment correction by OWHTO result in PF compartment offloading and should be considered when identifying the surgical indications for OWHTO. LEVEL OF EVIDENCE: Therapeutic, Level IV.

Early medial reconstruction combined with severely injured medial collateral ligaments can decrease residual medial laxity in anterior cruciate ligament reconstruction.

INTRODUCTION: This study aimed to describe an anatomic medial knee reconstruction technique for combined anterior cruciate ligament (ACL) and grade III medial collateral ligament (MCL) injuries and to assess knee function and stability restoration in patients who underwent primary MCL reconstruction compared with primary repair. METHODS: A total of 105 patients who had undergone anatomic ACL reconstruction between 2008 and 2017 were enrolled in this retrospective study and divided into two groups according to concomitant MCL ruptures. Group A included patients with isolated ACL ruptures without MCL injuries. Group B included patients with both ACL and MCL injuries, and it was subdivided into three groups according to the severity of the MCL injury and treatment modality: B-1, grade I or II MCL injury treated conservatively; B-2: grade III MCL injury treated by primary MCL repair; and B-3: grade III MCL injury treated by primary reconstruction. Knee stability was measured via Telos valgus radiography at 6-month and 2-year postoperative. The Lysholm score, Tegner activity level, Likert scales (satisfaction), and return to previous sports were evaluated at 2-year postoperative. RESULTS: At 6-month postoperative, there was no significant difference in medial laxity between the B-2 and B-3 groups. However, at 2-year postoperative, medial laxity were significantly higher both at 30° of flexion (5.2° versus 2.2°, p = 0.020) and at full extension (3.4° versus 1.1°, p < 0.001) in patients in B-2 group compared to those in B-3 group. There were no statistically significant differences between the two groups with respect to Lysholm scores, Tegner activity levels, Likert scales (satisfaction), and returning to previous sports at the 2-year follow-up. CONCLUSION: Primary medial reconstruction combined with severely injured MCL in ACL reconstruction may decrease residual medial laxity more than primary repair. LEVEL OF EVIDENCE: Retrospective observational study, IV.

The deubiquitinating enzyme Usp14 controls ciliogenesis and Hedgehog signaling.

Primary cilia are hair-like organelles that play crucial roles in vertebrate development, organogenesis and when dysfunctional result in pleiotropic human genetic disorders called ciliopathies, characterized by overlapping phenotypes, such as renal and hepatic cysts, skeletal defects, retinal degeneration and central nervous system malformations. Primary cilia act as communication hubs to transfer extracellular signals into intracellular responses and are essential for Hedgehog (Hh) signal transduction in mammals. Despite the renewed interest in this ancient organelle of growing biomedical importance, the molecular mechanisms that trigger cilia formation, extension and ciliary signal transduction are still not fully understood. Here we provide, for the first time, evidence that the deubiquitinase ubiquitin-specific protease-14 (Usp14), a major regulator of the ubiquitin proteasome system (UPS), controls ciliogenesis, cilia elongation and Hh signal transduction. Moreover, we show that pharmacological inhibition of Usp14 positively affects Hh signal transduction in a model of autosomal dominant polycystic kidney disease. These findings provide new insight into the spectrum of action of UPS in cilia biology and may provide novel opportunities for therapeutic intervention in human conditions associated with ciliary dysfunction.

Femoral Tunnel Widening After Double-Bundle Anterior Cruciate Ligament Reconstruction With Hamstring Autograft Produces a Small Shift of the Tunnel Position in the Anterior and Distal Direction: Computed Tomography-Based Retrospective Cohort Analysis.

PURPOSE: To determine whether the femoral tunnel position remains in an anatomical footprint after tunnel widening and shifting. METHODS: Patients who underwent unilateral double-bundle anterior cruciate ligament reconstruction with hamstring autograft and performed computed tomography scan evaluation at the time of 5 days and 1 year postoperatively were included in this retrospective cohort study. Three-dimensional models of the femur and femoral tunnels were reconstructed from computed tomography scan data. The location of the tunnel center and tunnel margins in the anatomical coordinate system, and the mean shifting distance of tunnel center and margin were measured with image analysis software during the period. The change of tunnel center location in Bernard quadrant was confirmed if the tunnel center remained within the boundaries of anatomical position after tunnel widening. RESULTS: A total of 56 patients satisfied the inclusion criteria. The mean shifting distance of AM and PL tunnel centers were 1.7 ± 0.9 mm and 1.6 ± 0.6 mm. The Tunnel margin of the anteromedial (AM) and posteromedial (PL) tunnels were shifted to 2.5 ± 1.3 mm and 2.6 ± 1.4 mm in the anterior direction, and 1.4 ± 0.9 mm and 1.0 ± 0.7 mm in the distal direction, respectively. Among the anatomical located tunnel, 97% (32/33) and 87.1% (27/31) of AM and PL tunnel centers remained in a range of anatomical footprint. The tunnel center was shifted from the anatomical position into a nonanatomical position in 3% (1/33) of the AM tunnel and 12.9% (4/31) of PL tunnel after tunnel widening. The tunnel location which shifted nonanatomically were relatively anterior and distal position. CONCLUSIONS: Tunnel widening shifts the tunnel position to the anterior and distal direction, which could change the initial tunnel position. Nevertheless, the majority of tunnel positions remained in the anatomical position after tunnel widening and shifting. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

Small-Molecule Inhibitors Targeting Proteasome-Associated Deubiquitinases.

The 26S proteasome is the principal protease for regulated intracellular proteolysis. This multi-subunit complex is also pivotal for clearance of harmful proteins that are produced throughout the lifetime of eukaryotes. Recent structural and kinetic studies have revealed a multitude of conformational states of the proteasome in substrate-free and substrate-engaged forms. These conformational transitions demonstrate that proteasome is a highly dynamic machinery during substrate processing that can be also controlled by a number of proteasome-associated factors. Essentially, three distinct family of deubiquitinases-USP14, RPN11, and UCH37-are associated with the 19S regulatory particle of human proteasome. USP14 and UCH37 are capable of editing ubiquitin conjugates during the process of their dynamic engagement into the proteasome prior to the catalytic commitment. In contrast, RPN11-mediated deubiquitination is directly coupled to substrate degradation by sensing the proteasome's conformational switch into the commitment steps. Therefore, proteasome-bound deubiquitinases are likely to tailor the degradation events in accordance with substrate processing steps and for dynamic proteolysis outcomes. Recent chemical screening efforts have yielded highly selective small-molecule inhibitors for targeting proteasomal deubiquitinases, such as USP14 and RPN11. USP14 inhibitors, IU1 and its progeny, were found to promote the degradation of a subset of substrates probably by overriding USP14-imposed checkpoint on the proteasome. On the other hand, capzimin, a RPN11 inhibitor, stabilized the proteasome substrates and showed the anti-proliferative effects on cancer cells. It is highly conceivable that these specific inhibitors will aid to dissect the role of each deubiquitinase on the proteasome. Moreover, customized targeting of proteasome-associated deubiquitinases may also provide versatile therapeutic strategies for induced or repressed protein degradation depending on proteolytic demand and cellular context.

Protective effect of EX-527 against high-fat diet-induced diabetic nephropathy in Zucker rats.

High-fat diet (HFD)-induced obesity is implicated in diabetic nephropathy (DN). EX-527, a selective Sirtuin 1 (SIRT1) inhibitor, has multiple biological functions; however, its protective effect against DN is yet to be properly understood. This study was aimed to explore the protective effect of EX-527 against DN in HFD-induced diabetic Zucker (ZDF) rats. After 21 weeks of continually feeding HFD to the rats, the apparent characteristics of progressive DN were observed, which included an increase in kidney weight (~160%), hyperglycemia, oxidative stress, and inflammatory cytokines, and subsequent renal cell damage. However, the administration of EX-527 for 10 weeks significantly reduced the blood glucose concentration and kidney weight (~59%). Furthermore, EX-527 significantly reduced the serum concentration of transforming growth factor-ß1 (49%), interleukin (IL)-1ß (52%), and IL-6 in the HFD-fed rats. Overall, the antioxidant activities significantly increased, and oxidative damage to lipids or DNA was suppressed. Particularly, EX-527 significantly reduced blood urea nitrogen (81%), serum creatinine (71%), microalbumin (43%), and urinary excretion of protein-based biomarkers. Histopathological examination revealed expansion of the extracellular mesangial matrix and suppression of glomerulosclerosis following EX-527 administration. EX-527 downregulated the expression of α-SMA (~64%), TGF-ß (25%), vimentin, α-tubulin, fibronectin, and collagen-1 in the kidneys of the HFD-fed rats. Additionally, EX-527 substantially reduced claudin-1 and SIRT1 expression, but increased the expression of SIRT3 in the kidneys of the HFD-fed rats. EX-527 also inhibited the growth factor receptors, including EGFR, PDGFR-ß, and STAT3, which are responsible for the anti-fibrotic effect of SIRT-1. Therefore, the administration of EX-527 protects against HFD-induced DN.

Preoperative latent medial laxity and correction angle are crucial factors for overcorrection in medial open-wedge high tibial osteotomy.

PURPOSE: This study aimed to determine which preoperative factors affect the postoperative change in the joint line convergence angle (JLCA) by preoperatively quantifying soft tissue laxity. METHODS: Thirty-four patients who underwent medial open-wedge high tibial osteotomy (HTO) with a navigation were analysed. The JLCA change after HTO was calculated using standing long-bone anteroposterior radiographs taken preoperatively and 6 months postoperatively. Latent soft tissue laxity was defined as the amount of soft tissue that can be extended to valgus or varus from the weight-bearing position, and calculated by subtracting the JLCA on weight-bearing standing radiographs from that on stress radiographs. Multiple linear regression was performed to determine the preoperative factors that statistically correlated with the postoperative JLCA change. RESULTS: In multiple linear regression, JLCA change had a statistically significant correlation with latent medial laxity (R = 0.6) and a statistically borderline significant correlation with correction angle (R = 0.2). These imply that the postoperative JLCA change increased by 0.6° per 1° increase in latent medial laxity, and increased by 0.2° per 1° increase in correction angle. Latent medial laxity was the most crucial factor associated with postoperative JLCA changes. CONCLUSION: The JLCA change could be larger in patients with large latent medial laxity or severe varus deformity requiring a large correction, which could lead to unexpected overcorrection in HTO. Postoperative JLCA change should be considered in preoperative surgical planning. Target point shifting within the hypomochlion point could be a strategy to prevent overcorrection, especially in patients with large latent medial laxity. LEVEL OF EVIDENCE: Level IV.

Deubiquitination Reactions on the Proteasome for Proteasome Versatility.

The 26S proteasome, a master player in proteolysis, is the most complex and meticulously contextured protease in eukaryotic cells. While capable of hosting thousands of discrete substrates due to the selective recognition of ubiquitin tags, this protease complex is also dynamically checked through diverse regulatory mechanisms. The proteasome's versatility ensures precise control over active proteolysis, yet prevents runaway or futile degradation of many essential cellular proteins. Among the multi-layered processes regulating the proteasome's proteolysis, deubiquitination reactions are prominent because they not only recycle ubiquitins, but also impose a critical checkpoint for substrate degradation on the proteasome. Of note, three distinct classes of deubiquitinating enzymes-USP14, RPN11, and UCH37-are associated with the 19S subunits of the human proteasome. Recent biochemical and structural studies suggest that these enzymes exert dynamic influence over proteasome output with limited redundancy, and at times act in opposition. Such distinct activities occur spatially on the proteasome, temporally through substrate processing, and differentially for ubiquitin topology. Therefore, deubiquitinating enzymes on the proteasome may fine-tune the degradation depending on various cellular contexts and for dynamic proteolysis outcomes. Given that the proteasome is among the most important drug targets, the biology of proteasome-associated deubiquitination should be further elucidated for its potential targeting in human diseases.

Activation of SIRT1 by L-serine increases fatty acid oxidation and reverses insulin resistance in C2C12 myotubes.

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, and the function is linked to cellular metabolism including mitochondrial biogenesis. Hepatic L-serine concentration is decreased significantly in fatty liver disease. We reported that the supplementation of the amino acid ameliorated the alcoholic fatty liver by enhancing L-serine-dependent homocysteine metabolism. In this study, we hypothesized that the metabolic production of NAD+ from L-serine and thus activation of SIRT1 contribute to the action of L-serine. To this end, we evaluated the effects of L-serine on SIRT1 activity and mitochondria biogenesis in C2C12 myotubes. L-Serine increased intracellular NAD+ content and led to the activation of SIRT1 as determined by p53 luciferase assay and western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) acetylation. L-Serine treatment increased the expression of the genes associated with mitochondrial biogenesis and enhanced mitochondrial mass and function. In addition, L-serine reversed cellular insulin resistance determined by insulin-induced phosphorylation of Akt and GLUT4 expression and membrane translocation. L-Serine-induced mitochondrial gene expression, fatty acid oxidation, and insulin sensitization were mediated by enhanced SIRT1 activity, which was verified by selective SIRT1 inhibitor (Ex-527) and siRNA directed to SIRT1. L-Serine effect on cellular NAD+ level is dependent on the L-serine metabolism to pyruvate that is subsequently converted to lactate by lactate dehydrogenase. In summary, these data suggest that L-serine increases cellular NAD+ level and thus SIRT1 activity in C2C12 myotubes.

Correction to: Activation of SIRT1 by L-serine increases fatty acid oxidation and reverses insulin resistance in C2C12 myotubes.

AbstractThe original version of this article unfortunately contained a mistake in the article title.

Z-ligustilide and n-Butylidenephthalide Isolated from the Aerial Parts of Angelica tenuissima Inhibit Lipid Accumulation In Vitro and In Vivo.

Abnormal lipid metabolism, such as increased fatty acid uptake and esterification, is associated with nonalcoholic fatty liver disease (NAFLD). The aqueous extract of the aerial part of Angelica tenuissima Nakai (ATX) inhibited high-fat diet-induced hepatic steatosis in mice as well as oleic acid-induced neutral lipid accumulation in HepG2 cells. ATX decreased the mRNA and protein levels of CD36 and diglyceride acyltransferase 2 (DGAT2), the maturation of sterol regulatory element-binding proteins (SREBP), and the expression of the lipogenic target genes fasn and scd1. The ATX components, Z-ligustilide and n-butylidenephthalide, inhibited the expression of FATP5 and DGAT2 and thus oleic acid-induced lipid accumulation in HepG2 cells. These results suggest that ATX and its active components Z-ligustilide and n-butylidenephthalide inhibit fatty acid uptake and esterification in mice and have potential as therapeutics for NAFLD.

Structural basis for dynamic regulation of the human 26S proteasome.

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near-atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

Predictive Factors for Patellofemoral Degenerative Progression After Opening-Wedge High Tibial Osteotomy.

PURPOSE: To identify risk factors for patellofemoral degenerative progression after opening-wedge high tibial osteotomy (HTO) and to investigate the effect of patellofemoral degeneration on the patellofemoral specific patient-reported outcomes. METHODS: Between March 2010 and June 2016, 94 knees (86 patients) underwent hardware removal with second-look arthroscopy at 21.4 months after opening-wedge HTO with first-look arthroscopy (mean follow-up duration, 49.8 months). Predictive factors for patellofemoral degeneration, including demographics, preoperative and postoperative mechanical axis (MA) of the lower limb (positive and negative MA indicating varus and valgus, respectively), tibial slope, and modified Blackburne-Peel ratio, were evaluated. Patients were divided into the progression and nonprogression groups according to their patellofemoral degenerative progression from first to second arthroscopy. Clinical outcomes, including the Kujala score and Knee Injury and Osteoarthritis Outcome Score, and radiographic outcomes were compared between the 2 groups. RESULTS: Postoperative MA (adjusted odd ratio, 0.62; P < .001) was the most significant predictive factor for progressive change in the patellofemoral joint (Rn2 = 0.31). Twenty-eight knees (30%) showed patellofemoral degenerative progression. Mean postoperative Kujala score (progression group 60.5 vs nonprogression group, 72.3; P = .005) and Knee Injury and Osteoarthritis Outcome Score scales (except for the symptom subscale) were lower in the progression group. Postoperative MA was significantly more corrected in the progression group (progression group -5.1° ± 2.7° vs nonprogression group -2.4° ± 2.3°; P < .001). CONCLUSIONS: Postoperative MA, which might be related to overcorrection, is correlated with patellofemoral degenerative progression after opening-wedge HTO. Patients with patellofemoral degenerative progression showed inferior patient-reported outcomes. LEVEL OF EVIDENCE: Level IV, case series with subgroup analysis.

Radiological grading of osteoarthritis on Rosenberg view has a significant correlation with clinical outcomes after medial open-wedge high-tibial osteotomy.

PURPOSE: To determine the classification scheme for osteoarthritis severity grading that most closely correlates with postoperative clinical outcomes and to identify the positive and negative prognostic factors for medial open-wedge high-tibial osteotomy (OWHTO). METHODS: Seventy-nine consecutive patients with primary varus osteoarthritis were treated using OWHTO. Arthritic grading was determined by arthroscopic assessment according to the modified Outerbridge classification and by radiographic classification according to the Kellgren-Lawrence (KL) grading scale on standing anteroposterior (AP) and 45° posteroanterior (PA) flexion weight-bearing radiography. Clinical outcome was assessed using the Oxford Knee Score (OKS), which was evaluated both preoperatively and at the postoperative 2-year follow-up after OWHTO. Multivariate regression analyses were used to explore and quantify the influence of baseline patient demographics, variables related to arthroscopic and radiological grades of arthritis, as well as postoperative alignment changes on the OKS. RESULTS: At the 2-year follow-up, the mean OKS had improved from 20 ± 4 to 39 ± 5 points (p < 0.001). The average mechanical femorotibial and mechanical medial proximal tibial angle (MPTA) changed from 6.9° ± 3.4° to valgus 2.7° ± 2.8° and from 85.6° ± 2.4° to 92.9° ± 3.7° (all p < 0.001). The osteoarthritis severity grade based on the KL scale was 2.4 ± 0.9 on standing AP radiography, 2.8 ± 0.9 on 45° PA flexion weight-bearing radiography (p = 0.003), and 3.4 ± 0.7 according to the modified Outerbridge classification. In the multivariate analyses, the KL grade on 45° PA flexion weight-bearing radiography (p = 0.01) and postoperative MPTA (p = 0.01) showed significant negative correlations with postoperative OKS at the 2-year follow-up. CONCLUSION: The KL grading system based on 45° PA flexion weight-bearing radiography showed the strongest significant negative correlation with postoperative OKS after the OWHTO procedure using three different common OA classification schemes, which should be considered to determine the surgical indication of HTO. The KL grading system based on 45° PA flexion weight-bearing radiography showed the strongest correlation with high-tibial osteotomy-surgical indications and the counselling of patients with advanced osteoarthritis. LEVEL OF EVIDENCE: IV.

An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau elimination in cultured neurons.

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.

Structure of proteasome ubiquitin receptor hRpn13 and its activation by the scaffolding protein hRpn2.

Rpn13 is a subunit of the proteasome that serves as a receptor for both ubiquitin and Uch37, one of the proteasome's three deubiquitinating enzymes. We have determined the structure of full-length human Rpn13 (hRpn13). Unexpectedly, Rpn13's ubiquitin- and Uch37-binding domains pack against each other when it is not incorporated into the proteasome. This intramolecular interaction reduces hRpn13's affinity for ubiquitin. We find that hRpn13 binding to the proteasome scaffolding protein hRpn2/S1 abrogates its interdomain interactions, thus activating hRpn13 for ubiquitin binding. hRpn13's Uch37-binding domain, a previously unknown fold, contains nine alpha helices. We have mapped its Uch37-binding surface to a region rich in charged amino acids. Altogether, our results provide mechanistic insights into hRpn13's functional activities with Uch37 and ubiquitin and suggest that its role as a ubiquitin receptor is finely tuned for proteasome targeting.