Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control.

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and, subsequently, CDK5RAP3, an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation, the E3 complex associated with ufmylated RPL26 on the 60S subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results provide insights into not only the molecular basis of the ufmylation but also its role in proteostasis.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex.

In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.

The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.

Loss of Atg2b and Gskip Impairs the Maintenance of the Hematopoietic Stem Cell Pool Size.

A germ line copy number duplication of chromosome 14q32, which contains ATG2B and GSKIP, was identified in families with myeloproliferative neoplasm (MPN). Here, we show that mice lacking both Atg2b and Gskip, but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of ATG2B and GSKIP, the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double-knockout fetal livers was significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of Atg2b or Gskip alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that Atg2b and Gskip play a synergistic effect in maintaining the pool size of HSCs.

Phase-separated protein droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show reduced inner fluidity.

Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid-liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget's disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the antioxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein-protein interaction assay, doxycycline-inducible gene expression system, and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.

Impaired osteoblastic behavior and function on saliva-contaminated titanium and its restoration by UV treatment.

The objective of this study was to examine behavior and function of osteoblasts on saliva-contaminated titanium and its potential improvement after UV light treatment. Acid-etched titanium disks were contaminated with human saliva. Osteoblasts derived from rat femur were cultured on contaminated and clean titanium disks. Contaminated disks further treated with UV light were also tested. The number of attached cells, the degree of cell spreading, and the expression of adhesion protein were significantly decreased on saliva-contaminated surfaces compared with clean surfaces. The gene expression of osteocalcin was also downregulated on contaminated surfaces, whereas ALP activity and mineralization were not significantly influenced. The impaired functions on contaminated surfaces were significantly increased if the surfaces were further treated with UV and even outperformed the ones on clean titanium surfaces. XPS analysis revealed that the atomic percentage of carbon and nitrogen detected on contaminated surfaces were substantially decreased after UV treatment. These results suggest that osteoblastic behavior and function were compromised on titanium surfaces contaminated with saliva. The compromised functions no longer happened if the surfaces were further treated with UV light, providing the basis to understand the effect of biological contamination on osseointegration and to explore UV treatment as a decontaminating technology.

Associations between dietary n-6 and n-3 fatty acids and arachidonic acid compositions in plasma and erythrocytes in young and elderly Japanese volunteers.

BACKGROUND: We reported that the compositions of arachidonic acid (ARA) in erythrocytes and plasma phospholipids (PL) in the elderly were lower than those in the young, though the ARA intake was nearly identical. OBJECTIVE: We further analyzed data in four study groups with different ages and sexes, and determined that the blood ARA levels were affected by the kinds of dietary fatty acids ingested. METHODS: One hundred and four healthy young and elderly volunteers were recruited. Dietary records together with photographic records from 28 consecutive days were reviewed and the fatty acid composition in plasma lipid fractions and erythrocyte PL was analyzed. RESULTS: No correlations for ARA between dietary fatty acids and blood lipid fractions were observed. A significant negative correlation between eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) intake and ARA composition in erythrocyte PL was observed. ARA composition in erythrocyte PL was significantly lower in elderly subjects than in young subjects, because EPA and DHA intake in elderly subjects was higher than in young subjects. However, after removing the effect of dietary EPA+DHA intake, the ARA composition in erythrocyte PL in elderly subjects was significantly lower than that in young subjects. CONCLUSIONS: Changes in physical conditions with aging influenced the low ARA composition of erythrocyte in elderly subjects in addition to the effects of dietary EPA and DHA.

Age-related changes of dietary intake and blood eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid levels in Japanese men and women.

We studied the relationship between dietary intake and the blood compositions of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA) in four study groups with different ages and sexes. One hundred and four subjects were recruited. Dietary records together with photographic records from 28 consecutive days were amassed and the fatty acid composition in erythrocyte membranes and plasma lipid fractions was analyzed. Fish intake in the elderly group was significantly higher than that in the young group in both men and women. The compositions of ARA in erythrocytes and plasma phospholipids in the elderly were lower than those in the young, but the ARA intake was nearly identical. In the elderly group, the percentage of dietary ARA consumed at the same time as EPA and DHA derived from fish was high. We considered that these fatty acids markedly inhibited the incorporation of dietary ARA into blood phospholipids.

Low-dose arachidonic acid intake increases erythrocytes and plasma arachidonic acid in young women.

Arachidonic acid (ARA) is considered to be a minor contributor to the diet. Previous reports regarding the effect of ARA supplementation on the composition of long-chain polyunsaturated fatty acids (LCPUFA) in the blood of humans are extremely limited. In the present study, we conducted a crossover double-blind, placebo-control study. Twenty-three young Japanese women consumed one capsule containing triacylglycerol enriched with 80 mg ARA, equivalent to the amount in one egg, daily for 3 weeks. Blood samples were drawn before and after treatment periods, and the compositions of the LCPUFA in blood lipid fractions were measured. The supplementation of ARA increased the composition of ARA, but did not decrease the composition of n-3LCPUFA in erythrocyte phospholipids and plasma phospholipids, esterified cholesterol, and triacylglycerol. We found that dietary ARA increased the ARA level in all lipid fractions of the blood, even at a very low dose.

Trigger point acupuncture for treatment of knee osteoarthritis--a preliminary RCT for a pragmatic trial.

BACKGROUND: There is evidence for the efficacy of acupuncture treatment in knee osteoarthritis, but it remains unclear which acupuncture modes are most effective. We evaluated the effects of trigger point acupuncture on pain and quality of life in knee osteoarthritis patients, compared with acupuncture at standard points, and sham acupuncture. METHODS: Thirty patients (27 women, 3 men; aged 61-82 years) with non-radiating knee osteoarthritis pain for at least six months and normal neurological examination were randomised to one of three groups for the study period of 21 weeks. Each group received five acupuncture treatment sessions. The standard acupuncture point group (n=10) received treatment at traditional acupuncture points for knee pain; the trigger point acupuncture group (n=10) received treatment at trigger points; and the third group (n=10) received sham acupuncture treatment at the trigger points. Outcome measures were pain intensity (visual analogue scale, VAS) and WOMAC index (Western Ontario and McMaster Universities Arthritis Index). The groups were compared by the area under the curve method. RESULTS: Five patients dropped out of the study because of lack of improvement, and one patient (in the trigger point acupuncture group) dropped out because of deterioration of symptoms; the remaining 24 patients were included in the analysis. After treatment, the trigger point acupuncture group reported less pain intensity on VAS than the standard acupuncture or sham treatment group, but both the trigger point acupuncture and standard acupuncture groups reported improvement of function of knee. There was a significant reduction in pain intensity between pre-treatment and five weeks after treatment for the trigger point acupuncture (P<0.01) and standard acupuncture groups (P<0.01) included in the analysis, but not for the sham treatment group. Group comparison using the area under the curves demonstrated a significant difference only between trigger point acupuncture and sham treatment groups analysed (P<0.025 for VAS, and P<0.031 for WOMAC). CONCLUSION: These results suggest that trigger point acupuncture therapy may be more effective for osteoarthritis of the knee in some elderly patients than standard acupuncture therapy.

A pilot study on using acupuncture and transcutaneous electrical nerve stimulation (TENS) to treat knee osteoarthritis (OA).

BACKGROUND: The present study tests whether a combined treatment of acupuncture and transcutaneous electrical nerve stimulation (TENS) is more effective than acupuncture or TENS alone for treating knee osteoarthritis (OA). METHODS: Thirty-two patients with knee OA were randomly allocated to four groups. The acupuncture group (ACP) received only acupuncture treatment at selected acupoints for knee pain; the TENS group (TENS) received only TENS treatment at pain areas; the acupuncture and TENS group (A&T) received both acupuncture and TENS treatments; the control group (CT) received topical poultice (only when necessary). Each group received specific weekly treatment five times during the study. Outcome measures were pain intensity in a visual analogue scale (VAS) and knee function in terms of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). RESULTS: The ACP, TENS and A&T groups reported lower VAS and WOMAC scores than the control group. Significant reduction in pain intensity (P = 0.039) and significant improvement in knee function (P = 0.008) were shown in the A&T group. CONCLUSION: Combined acupuncture and TENS treatment was effective in pain relief and knee function improvement for the sampled patients suffering from knee OA.

Randomised trial of trigger point acupuncture compared with other acupuncture for treatment of chronic neck pain.

INTRODUCTION: There is some evidence for the efficacy of acupuncture in chronic neck pain (CNP) treatment, but it remains unclear which acupuncture modes are most effective. Objective was to evaluate the effects of trigger point acupuncture on pain and quality of life (QOL) in CNP patients compared to three other acupuncture treatments (acupoints, non-trigger point and sham treatment). METHODS: Forty out-patients (29 women, 11 men; age range: 47-80 years) from the Department of Orthopaedic Surgery, Meiji University of Oriental Medicine, with non-radiating CNP for at least 6 months and normal neurological examination were randomised to one of four groups over 13 weeks. Each group received two phases of acupuncture treatment with an interval between them. The acupoint group (standard acupuncture; SA, n=10) received treatment at traditional acupoints for neck pain, the trigger point (TrP, n=10) and non-trigger point (non-TrP, n=10) groups received treatment at tenderness points for the same muscle, while the other acupuncture group received sham treatments on the trigger point (SH, n=10). Outcome measures were pain intensity (visual analogue scale; VAS 0-100mm) and disease specific questionnaire (neck disability index; NDI, 60-point scale). RESULTS: After treatment, the TrP group reported less pain intensity and improved QOL compared to the SA or non-TrP group. There was significant reduction in pain intensity between the treatment and the interval for the TrP group (p<0.01, Dunnett's multiple test), but not for the SA or non-TrP group. CONCLUSION: These results suggest that trigger point acupuncture therapy may be more effective on chronic neck pain in aged patients than the standard acupuncture therapy.

[An elderly woman with duodenal perforation difficulty diagnose].

An 86-year-old woman was referred with acute epigastric pain. She had tenderness, but no muscular guarding of the epigastric lesion. Abdominal ultrasound showed a gallstone with a normal gallbladder wall and no ascites. The white blood cell count was 11,600/mm(3), but she was negative for C-reactive protein (CRP). An upper gastrointestinal tract endoscopic examination revealed only edema of the duodenal mucosa. Although H2-receptor antagonists were given, she had to be admitted due to chills and high fever. While the abdominal symptoms did not change, the CRP concentration became 14.79mg/dl. While plain abdominal X-ray did not show an abnormal gas pattern, subsequent abdominal CT examination showed air and fluid collection around the second portion of the duodenum. We diagnosed duodenal perforation and prepared for emergency operation. However, her general condition had markedly deteriorated during the hours. Laparotomy revealed a free purulent fluid around second portion of the duodenum caused by perforation of a duodenal diverticulum. The patient gradually recovered and was discharged after 58 days. Since a duodenal perforation in an elderly patient is difficult to diagnose early in spite of serious illness, abdominal CT should be encouraged.

Effects of trigger point acupuncture on chronic low back pain in elderly patients--a sham-controlled randomised trial.

INTRODUCTION: There is some evidence for the efficacy of acupuncture, but it remains unclear whether trigger point acupuncture is effective. Our objective was to evaluate the effects of trigger point acupuncture on pain and quality of life in chronic low back pain patients compared with sham acupuncture. METHODS: Twenty-six consecutive out-patients (17 women, 9 men; age range: 65-91 years) from the Department of Orthopaedic Surgery, Meiji University of Oriental Medicine, with non-radiating low back pain for at least six months and normal neurological examination, were randomised to two groups. Each group received one phase of trigger point acupuncture and one of sham acupuncture with a three week washout period between them, over 12 weeks. Group A (n = 13) received trigger point acupuncture in the first phase and sham acupuncture in the second. Group B (n = 13) received the same interventions in the reverse order. Outcome measures were pain intensity (visual analogue scale, VAS) and Roland Morris Questionnaire. RESULTS: Nineteen patients were included in the analysis. At the end of the first treatment phase, group A receiving trigger point acupuncture scored significantly lower VAS (P < 0.001) and Roland Morris Questionnaire scores (P < 0.01) than the sham control group. There were significant within-group reductions in pain in both groups during the trigger point acupuncture phase but not in the sham treatment phase. However, the beneficial effects were not sustained. CONCLUSION: These results suggest that trigger point acupuncture may have greater short term effects on low back pain in elderly patients than sham acupuncture.

Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues.

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L(1) to S(37)) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K(15) to V(32)) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14(+) murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14(+) cells and attenuating production of tumor necrosis factor alpha (TNF-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.