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Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions., (© 2021. The Author(s).)

KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7-3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.

Background: Relatively few studies investigated the association between rest-activity circadian rhythm and cognitive impairment in population-based study, and the evidence from Asian populations is sparse. We aimed to examine the relationship of actigraphy measured rest-activity circadian rhythm with mild cognitive impairment (MCI) or cognitive impairment in Hong Kong healthy community-dwelling older adults., Methods: We recruited 174 Hong Kong healthy adults aged ≥65 years (36 male vs. 138 female) during April-September 2018, and followed up them for 12 months. Participants were invited to wear wrist actigraphy for 7 days in both baseline and follow-up study. We used the actigraph data to calculate their midline statistic of rhythm (MESOR), amplitude, acrophase and percent rhythm. Montreal Cognitive Assessment (MoCA) was used to assess their cognitive scores at baseline and follow-up. Multivariate logistic regression model was performed to estimate the association of rest-activity circadian rhythm parameters with MCI; whilst multinomial logistic regression model was used to examine the association between rhythm parameters and changes of cognitive scores (i.e., worsen: <-1, stable: -1 to 1, better cognition: ≥2) after 12-months follow-up respectively., Results: There was no association between rest-activity circadian rhythm parameters and MCI or cognitive impairment at baseline. Compared to those with an averaged value of acrophase (1:24pm-3:00pm), results of multinominal logistic regression showed that participants with a delayed acrophase (after 3:00pm) were less likely to have better cognition (adjusted odds ratio (AOR) = 0.32, 95% confidence interval (CI) = 0.11-0.88). Upon one year of follow-up, participants who delayed their acrophase for 24 min than their baseline measurements were also less likely to have better cognitive functions (AOR = 0.26, 95%CI = 0.08-0.79)., Conclusions: Results from both the baseline survey and follow-up study consistently confirmed that older adults, especially in light of the majority of participants being the females, with delayed acrophase were less likely to have better cognition in the Asian population., Competing Interests: None.Table A1Cutoff scores of changed of rest-activity circadian rhythm parameters after 12 monthsTable A1Changed of rest-activity circadian rhythm parameters 1Decrease/AdvancedStableIncreased/DelayedMESOR≤-31.62-32.62-15.84>15.84Amplitude≤-22.84-22.84-5.23>5.23Acrophase≤-36 min-36-24 min>24 minPercent rhythm≤-2.12-2.12-0.74>0.741All changes of rest-activity rhythms parameters were stratified based on their tertile distribution.Table A2Cutoff scores of The Hong Kong Cantonese version of Montreal Cognitive AssessmentTable A2AgeEducation (years)Cutoff percentile7th2nd65–690–31494–618137–9191610–122017>12232170–790–314114–615107–9181510–121918>122016≥800–61310>61513Wong A, Law LS, Liu W, Wang Z, Lo ES, Lau A, et al. Montreal Cognitive Assessment: One Cutoff Never Fits All. Stroke. 2015;46(12):3547-50.Table A3Odds ratio and 95% confidence interval of the association between selective characteristics and prevalent mild cognitive impairment or cognitive impairment at the baselineTable A3CharacteristicsCrude OR (95%CI)Adjusted OR (95%CI) 2Age1.06 (1.01–1.11)1.04 (0.98–1.10)Sex Male1.001.00 Female4.13 (1.53–14.48)3.52 (1.23–12.74)Educational attainment Never educated4.79 (2.29–10.23)2.65 (1.63–8.35) Educated1.001.00Marital status Married1.001.00 Single/Divorce/Widow1.39 (0.72–2.69)0.78 (0.36–1.63)Body mass index < 251.001.00 ≥ 251.27 (0.63–2.54)1.09 (0.50–2.35)Smoking Non-smoker1.001.00 Current/Ex-smoker1.01 (0.31–2.88)1.85 (0.46–7.46)Habitual alcohol consumption No1.001.00 Yes1.80 (0.51–5.93)2.23 (0.55–8.97)History of hypertension or diabetes No1.001.00 Yes1.06 (0.55–2.09)0.86 (0.42–1.78)Geriatric Depression Scale Non depressed (315.695 (33.3)1 (14.3)1.20 (0.06–24.47)3.08 (0.08–12.22)1 (14.3)0.30 (0.03–3.63)1.02 (0.05–21.23)19 (33.9)10 (34.5)1.05 (0.40–2.95)1.46 (0.43–4.92)10 (35.7)1.50 (0.48–4.76)1.43 (0.41–5.00)Amplitude ≤109.605 (33.3)2 (28.6)1.001.005 (71.4)1.001.0017 (30.4)9 (31.0)1.001.009 (32.1)1.001.00 109.60- 168.785 (33.3)3 (42.8)1.50 (0.17–13.23)0.36 (0.02–7.59)0 (0.0)––17 (30.4)11 (37.9)1.22 (0.40–3.70)1.46 (0.45–4.70)12 (42.9)1.33 (0.45–3.99)1.20 (0.39–3.73) >168.785 (33.3)2 (28.6)1.00 (0.01–10.17)0.87 (0.03–2.19)2 (28.6)0.40 (0.05–3.12)0.92 (0.05–1.82)22 (39.3)9 (31.0)0.77 (0.25–2.37)1.01 (0.29–3.52)7 (25.0)0.60 (0.19–1.94)0.50 (0.14–1.78)Acrophase ≤1:24pm2 (13.3)3 (42.9)1.50 (0.11–21.32)1.00 (0.05–2.19)3 (42.9)0.75 (0.06–8.83)0.56 (0.03–9.63)19 (33.9)12 (41.4)0.94 (0.32–2.78)0.95 (0.32–2.82)8 (28.6)0.57 (0.18–1.79)0.57 (0.18–1.79) 1:24pm- 3:00pm2 (13.3)2 (28.6)1.001.004 (57.1)1.001.0015 (26.8)10 (34.5)1.001.0011 (39.3)1.001.00 >3:00pm11 (73.3)2 (27.6)0.18 (0.02–2.15)0.11 (0.00–2.98)0 (0.0)––22 (39.3)7 (24.1)0.48 (0.15–1.53)0.49 (0.15–1.59)9 (32.1)0.56 (0.19–1.67)0.55 (0.18–1.66)Percent rhythm ≤8.166 (40.0)4 (57.1)1.001.004 (57.1)1.001.0018 (32.1)10 (34.5)1.001.007 (25.0)1.001.00 8.16–12.283 (20.0)1 (14.3)0.50 (0.04–6.69)0.52 (0.03–9.21)3 (42.9)1.50 (0.20–1.15)2.22 (0.21–2.34)17 (30.4)11 (37.9)1.16 (0.39–3.44)1.15 (0.39–3.41)9 (32.1)1.36 (0.41–4.48)1.37 (0.42–4.52) >12.286 (40.0)2 (28.6)0.50 (0.07–3.85)0.04 (0.24–5.97)0 (0.0)––21 (37.5)8 (27.6)0.69 (0.22–2.11)0.73 (0.23–2.26)12 (42.9)1.47 (0.48–4.53)1.45 (0.47–4.49)Abbreviations: OR, odds ratio; CI, confidence intervals, MESOR = Midline statistic of rhythm.1 Adjusted for the age at interview and education attainment in years.2 All rest-activity rhythms parameters were stratified based on their tertile distribution.Table A7Odds ratio and 95% confidence interval of the association between changes of rest-activity circadian rhythm and changed cognitive scores between baseline and follow-up measurements stratified by sexTable A7MaleFemaleChanges of rest-activity rhythms parameters 2Stable n = 71Worsen n = 36Better n = 35Stable n = 71Worsen n = 36Better n = 35N (%)N (%)Crude OR (95%CI)Adjusted OR (95%CI) 2N (%)Crude OR (95%CI)Adjusted OR (95%CI) 2N (%)N (%)Crude OR (95%CI)Adjusted OR (95%CI) 2N (%)Crude OR (95%CI)Adjusted OR (95%CI) 2MESOR ≤-31.622 (14.3)4 (57.1)––3 (42.9)2.50 (0.25–24.72)3.25 (0.24–44.41)17 (32.7)8 (30.8)0.94 (0.29–3.00)1.02 (0.31–3.30)10 (38.4)1.32 (0.34–3.30)1.35 (0.43–4.26) -32.62-15.845 (35.7)0 (0.0)––3 (42.9)1.001.0018 (34.6)9 (34.6)1.001.008 (30.8)1.001.00 >15.847 (50.0)3 (42.9)––1 (14.2)0.24 (0.02–3.01)0.32 (0.02–4.82)17 (32.7)9 (34.6)1.06 (0.42–4.15)0.98 (0.31–3.10)8 (30.8)1.05 (0.32–3.46)1.03 (0.31–3.42)Amplitude ≤-22.845 (35.7)3 (42.9)1.80 (0.12–26.20)1.89 (0.08–4.21)3 (42.9)1.80 (0.12–26.20)1.75 (0.10–30.93)15 (28.8)10 (38.5)1.20 (0.39–3.65)1.26 (0.40–4.03)8 (30.8)0.96 (0.30–3.05)0.93 (0.28–3.09) -22.84-5.233 (21.4)1 (14.2)1.001.001 (14.2)1.001.0018 (34.6)10 (38.5)1.001.0010 (38.4)1.001.00 >5.236 (42.9)3 (42.9)1.50 (0.12–21.31)1.96 (0.08–4.55)3 (42.9)1.50 (0.12–21.31)2.19 (0.12–41.70)19 (36.5)6 (23.1)0.57 (0.17–1.89)0.60 (0.18–2.08)8 (30.8)0.76 (0.24–2.35)0.74 (0.23–2.39)Acrophase ≤-36 min6 (42.9)1 (14.3)0.17 (0.01–2.09)0.23 (0.01–4.10)3 (42.9)2.00 (0.15–26.74)3.77 (0.18–79.49)18 (34.6)6 (23.1)0.72 (0.19–2.75)0.57 (0.14–2.31)8 (30.8)0.39 (0.13–1.18)0.35 (0.11–1.15) -36-24 min4 (28.6)4 (57.1)1.001.001 (14.3)1.001.0013 (25.0)6 (23.1)1.001.0015 (57.7)1.001.00 >24 min4 (28.6)2 (28.6)0.50 (0.06–4.47)1.21 (0.07–2.01)3 (42.9)3.00 (0.21–42.63)16.93 (0.52–553.16)21 (40.4)14 (53.8)1.44 (0.44–4.70)1.36 (0.41–4.49)3 (11.5)0.12 (0.03–0.51)0.12 (0.03–0.50)Percent rhythm ≤-2.124 (28.6)2 (28.6)1.00 (0.09–11.03)0.82 (0.05–1.46)2 (28.6)0.67 (0.07–6.41)0.37 (0.03–4.79)19 (36.5)14 (30.8)0.49 (0.16–1.52)0.47 (0.15–1.49)9 (34.6)0.83 (0.26–2.69)0.81 (0.25–2.67) -2.12-0.744 (28.6)2 (28.6)1.001.003 (42.9)1.001.0014 (26.9)12 (46.2)1.001.008 (30.8)1.001.00 >0.746 (42.9)3 (42.9)1.00 (0.11–8.95)0.80 (0.06–1.17)2 (28.6)0.44 (0.05–3.98)0.23 (0.02–3.09)19 (36.5)10 (23.1)0.36 (0.11–1.22)0.39 (0.16–1.31)9 (34.6)0.83 (0.26–2.69)0.84 (0.26–2.73)Abbreviations: OR, odds ratio; CI, confidence intervals, MESOR = Midline statistic of rhythm.1 Adjusted for the age at interview, sex, education attainment in years.2 All changes of rest-activity rhythms parameters were stratified based on their tertile distribution.Table A8Interrelationship between rest-activity circadian rhythm parameters and educational attainment.Table A8CharacteristicsMESORAmplitudeAcrophasePercent rhythmEducational attainment Never educated253.4 ± 68.79116.1 ± 48.0513:44 ± 2:3110.1 ± 6.41 Educated299.9 ± 78.24156.5 ± 61.5414:24 ± 2:1010.9 ± 5.42 Mean square67733.0051174.0013.9720.89 F-value11.6814.862.760.65 p-value<0.01<0.010.100.42Abbreviations: MESOR = Midline statistic of rhythmInterrelationship between rest-activity circadian rhythm parameters and educational attainment was assessed by one-way ANOVA.Table A9Odds ratio and 95% confidence interval of the association between baseline rest-activity circadian rhythm and changed cognitive scores during the follow-up stratified by educationTable A9Rest-activity rhythms parameters 2Stable n = 54Worsen n = 25Better n = 27N (%)N (%)Crude OR (95%CI)Adjusted OR (95%CI) 1N (%)Crude OR (95%CI)Adjusted OR (95%CI) 1MESOR ≤244.2615 (27.8)6 (24.0)1.001.009 (33.3)1.001.00 244.26- 315.6917 (31.5)12 (48.0)1.76 (0.53–5.87)1.79 (0.51–6.29)10 (37.0)0.98 (0.31–3.06)1.04 (0.32–3.44) >315.6922 (40.7)7 (28.0)0.80 (0.22–2.84)0.80 (0.20–3.19)8 (29.6)0.61 (0.19–1.93)0.65 (0.18–2.30)Amplitude ≤109.6011 (20.4)6 (24.0)1.001.009 (33.3)1.001.00 109.60- 168.7818 (33.3)12 (48.0)1.22 (0.36–4.20)1.13 (0.30–4.23)10 (37.0)0.68 (0.21–2.19)0.64 (0.18–2.24) >168.7825 (46.3)7 (28.0)0.51 (0.14–1.88)0.46 (0.11–1.98)8 (29.6)0.39 (0.11–1.28)0.38 (0.10–1.44)Acrophase ≤1:24pm16 (29.6)10 (40.0)0.86 (0.26–2.87)0.86 (0.26–2.88)7 (25.9)0.37 (0.11–1.23)0.36 (0.11–1.19) 1:24pm- 3:00pm11 (20.4)8 (32.0)1.001.0013 (48.1)1.001.00 >3:00pm27 (50.0)7 (28.0)0.36 (0.10–1.22)0.36 (0.10–1.22)7 (25.9)0.22 (0.07–0.70)0.22 (0.07–0.71)Percent rhythm ≤8.1618 (33.3)11 (44.0)1.001.007 (25.9)1.001.00 8.16–12.2814 (29.9)9 (36.0)1.05 (0.34–3.24)1.02 (0.33–3.18)9 (33.3)1.65 (0.49–5.54)1.66 (0.49–5.64) >12.2822 (40.7)5 (20.0)0.37 (0.11–1.27)0.35 (0.10–1.25)11 (40.7)1.29 (0.41–4.00)1.38 (0.43–4.45)Abbreviations: OR, odds ratio; CI, confidence intervals, MESOR = Midline statistic of rhythm.1 Adjusted for the age at interview and sex.2 All rest-activity rhythms parameters were stratified based on their tertile distribution.Table A10Odds ratio and 95% confidence interval of the association between changes of rest-activity circadian rhythm and changed cognitive scores between baseline and follow-up measurements stratified by education.Table A10Changes of rest-activity rhythms parameters 2Stable n = 50Worsen n = 23Better n = 25N (%)N (%)Crude OR (95%CI)Adjusted OR (95%CI) 1N (%)Crude OR (95%CI)Adjusted OR (95%CI) 1MESOR ≤-31.6215 (32.7)8 (30.8)1.51 (0.43–5.36)1.49 (0.41–5.38)9 (38.4)1.13 (0.36–3.60)1.31 (0.40–4.33) -32.62-15.8417 (34.6)6 (34.6)1.001.009 (30.8)1.001.00 >15.8418 (32.7)9 (34.6)1.42 (0.42–4.83)1.42 (0.41–4.87)7 (30.8)0.73 (0.22–2.41)0.75 (0.23–2.51)Amplitude ≤-22.8416 (32.0)10 (43.5)1.63 (0.44–5.96)1.58 (0.42–5.97)11 (44.0)1.28 (0.39–4.23)1.44 (0.42–4.94) -22.84-5.2313 (26.0)5 (21.7)1.001.007 (28.0)1.001.00 >5.2321 (42.0)8 (34.8)0.99 (0.27–3.69)0.95 (0.24–3.76)7 (28.0)0.62 (0.18–2.17)0.74 (0.20–2.73)Acrophase ≤-36 min20 (40.0)5 (21.7)0.50 (0.13–2.00)0.49 (0.12–2.01)8 (32.0)0.44 (0.13–1.39)0.36 (0.11–1.22) -36-24 min12 (24.0)6 (26.1)1.001.0011 (44.0)1.001.00 >24 min18 (36.0)12 (52.2)1.33 (0.39–4.53)1.33 (0.39–4.53)6 (24.0)0.36 (0.11–1.25)0.36 (0.10–1.26)Percent rhythm ≤-2.1219 (38.0)6 (26.1)0.42 (0.12–1.49)0.42 (0.12–1.50)11 (44.0)0.77 (0.25–2.41)0.72 (0.23–2.28) -2.12-0.7412 (24.0)9 (39.1)1.001.009 (36.0)1.001.00 >0.7419 (38.0)8 (34.7)0.56 (0.17–1.86)0.56 (0.17–1.87)5 (20.0)0.35 (0.09–1.30)0.33 (0.09–1.25)Abbreviations: OR, odds ratio; CI, confidence intervals, MESOR = Midline statistic of rhythm.1 Adjusted for the age at interview and sex.2 All changes of rest-activity rhythms parameters were stratified based on their tertile distribution., (© 2021 The Authors. Published by Elsevier Inc.)

Amputation of a salamander limb triggers a regeneration process that is perfect. A limited number of genes have been studied in this context and even fewer have been analyzed functionally. In this work, we use the BMP signaling inhibitor LDN193189 on Ambystoma mexicanum to explore the role of BMPs in regeneration. We find that BMP signaling is required for proper expression of various patterning genes and that its inhibition causes major defects in the regenerated limbs. Fgf8 is downregulated when BMP signaling is blocked, but ectopic injection of either human or axolotl protein did not rescue the defects. By administering LDN193189 treatments at different time points during regeneration, we show clearly that limb regeneration progresses in a proximal to distal fashion. This demonstrates that BMPs play a major role in patterning of regenerated limbs and that regeneration is a progressive process like development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

By using the Cre-mediated genetic switch technology, we were able to successfully generate a conditional knock-in mouse, bearing the KIF2A p.His321Asp missense point variant, identified in a subject with malformations of cortical development. These mice present with neuroanatomical anomalies and microcephaly associated with behavioral deficiencies and susceptibility to epilepsy, correlating with the described human phenotype. Using the flexibility of this model, we investigated RosaCre-, NestinCre- and NexCre-driven expression of the mutation to dissect the pathophysiological mechanisms underlying neurodevelopmental cortical abnormalities. We show that the expression of the p.His321Asp pathogenic variant increases apoptosis and causes abnormal multipolar to bipolar transition in newborn neurons, providing therefore insights to better understand cortical organization and brain growth defects that characterize KIF2A-related human disorders. We further demonstrate that the observed cellular phenotypes are likely to be linked to deficiency in the microtubule depolymerizing function of KIF2A., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair., Competing Interests: SZ, MP, FW, YL, LW, BB, TB, BK, AP No competing interests declared, (© 2020, Zhu et al.)

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m 7 G) cap on the 5' end of RNAs. The m 7 G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m 7 G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m 7 G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg- luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E., Competing Interests: The authors declare no competing interest.

Mutations in KIF14 have previously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells. KIF14 encodes a mitotic kinesin, which plays a key role at the midbody during cytokinesis and has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with fetuses presenting with the syndromic form and harbouring biallelic variants in KIF14. Our functional analyses showed that the identified variants severely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human fetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14 mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos, supporting a potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivo analyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggested that loss of kif14 causes ciliopathy-like phenotypes through an accumulation of mitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved function in cytokinesis during kidney and brain development., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mainstream partial nitritation/anammox (PN/A), coupled with excess biological phosphorus removal, in a 200,000 m 3 /d step-feed activated sludge process (Train 2) in the Changi Water Reclamation Plant (WRP), Singapore, has been studied and reported. This paper presents an overview of process performance and the microbial community during the period from 2011 to 2016. The site data showed that, along with the reduction of dissolved oxygen (DO) from 1.7 to 1.0 mg O 2 /L in the aeration zones, the concentrations of ammonium and nitrate of the final effluent increased, while nitrite decreased, resulting in an increase of 2.4 mg N/L of total inorganic nitrogen. Autotrophic nitrogen removal was higher than heterotrophic biological nitrogen removal under higher DO concentration conditions, but decreased under low DO operating condition. These macro-scale changes were caused by shifts of the nitrogen-converting microbial community. The ammonia oxidizing bacteria (AOB) population abundance was reduced by 30 times, while the nitrite oxidizing bacteria (NOB) population abundance and specific activity increased significantly with a shift of dominant genus from Nitrobacter to Nitrospira. The ratio of AOB and NOB specific activities were reduced from 12.8 to 1.6, and the ex situ nitrite accumulation ratio reduced from 76% to 29%. Changes in the microbial community and overall process performance illustrated that, compared to the excellent NOB suppression under high DO conditions, NOB were more active after the DO concentration reduction despite still being partly suppressed. This case study demonstrated, for the first time, the influence of DO reduction on the nitrogen conversion microbial community and PN/A process performance for a suspended growth system. Its relevance to biofilm and hybrid PN/A processes is also discussed.

Kinesin-13 proteins are major microtubule (MT) regulatory factors that catalyze removal of tubulin subunits from MT ends. The class-specific "neck" and loop 2 regions of these motors are required for MT depolymerization, but their contributing roles are still unresolved because their interactions with MT ends have not been observed directly. Here we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array, providing a view of these interactions. The neck of Kif2A binds to one tubulin dimer and the motor core to the other, guiding insertion of the KVD motif of loop 2 in between them. AMPPNP-bound Kif2A can form stable complexes with tubulin in solution and trigger MT depolymerization. We also demonstrate the importance of the neck in modulating ATP turnover and catalytic depolymerization of MTs. These results provide mechanistic insights into the catalytic cycles of kinesin-13.

Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear. Here, we show that in a depletion-and-rescue assay in Drosophila S2 cells, anillin lacking the entire actin-binding domain (ActBD) exhibits defective cortical localization during mitosis and a greatly diminished ability to support cytokinesis. Using in vitro binding assays and electron microscopy on recombinant fragments, we determine that the anillin ActBD harbors three distinct actin-binding sites (ABS 1-3). We show that each ABS binds to a distinct place on F-actin. Importantly, ABS1 and ABS3 partially overlap on the surface of actin and, therefore, interact with F-actin in a mutually exclusive fashion. Although ABS2 and ABS3 are sufficient for bundling, ABS1 contributes to the overall F-actin bundling activity of anillin and enables anillin to switch between two actin-bundling morphologies and promote the formation of three-dimensional F-actin bundles. Finally, we show that in live S2 cells, ABS2 and ABS3 are each required and together sufficient for the robust cortical localization of the ActBD during cytokinesis. Collectively, our structural, biochemical, and cell biological data suggest that multiple anillin-actin interaction modes promote the faithful progression of cytokinesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mainstream partial nitritation and Anammox (PN/A) has been observed and studied in the step-feed activated sludge process at the Changi water reclamation plant (WRP), which is the largest WRP (800,000 m 3 /d) in Singapore. This paper presents the study results for enhanced biological phosphorus removal (EBPR) co-existing with PN/A in the activated sludge process. Both the in-situ EBPR efficiency and ex-situ activities of phosphorus release and uptake were high. The phosphorus accumulating organisms were dominant, with little presence of glycogen accumulating organisms in the activated sludge. Chemical oxygen demand (COD) mass balance illustrated that the carbon usage for EBPR was the same as that for heterotrophic denitrification, owing to autotrophic PN/A conversions. This much lower carbon demand for nitrogen removal, compared to conventional biological nitrogen removal, made effective EBPR possible. This paper demonstrated for the first time the effective EBPR co-existence with PN/A in the mainstream in a large full-scale activated sludge process, and the feasibility to accommodate EBPR into the mainstream PN/A process. It also shows EBPR can work under warm climates.

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy., Competing Interests: The authors have declared that no conflict of interest exists.

Karyotype diversity is a hallmark of solid tumors that contributes to intratumor heterogeneity. This diversity is generated by persistent chromosome mis-segregation associated with chromosomal instability (CIN). CIN correlates with tumor relapse and is thought to promote drug resistance by creating a vast genomic landscape through which karyotypically unique clones survive lethal drug selection. We explore this proposition using a small molecule (UMK57) that suppresses chromosome mis-segregation in CIN cancer cells by potentiating the activity of the kinesin-13 protein MCAK. Sublethal doses of UMK57 destabilize kinetochore-microtubule (k-MT) attachments during mitosis to increase chromosome segregation fidelity. Surprisingly, chromosome mis-segregation rebounds in UMK57-treated cancer cells within a few days. This rapid relapse is driven by alterations in the Aurora B signaling pathway that hyper-stabilize k-MT attachments and is reversible following UMK57 removal. Thus, cancer cells display adaptive resistance to therapies targeting CIN through rapid and reversible changes to mitotic signaling networks., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks., (© 2014 Kachaner.)

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The mitotic kinesin motor protein KIF14 is essential for cytokinesis during cell division and has been implicated in cerebral development and a variety of human cancers. Here we show that the mouse KIF14 motor domain binds tightly to microtubules and does not display typical nucleotide-dependent changes in this affinity. It also has robust ATPase activity but very slow motility. A crystal structure of the ADP-bound form of the KIF14 motor domain reveals a dramatically opened ATP-binding pocket, as if ready to exchange its bound ADP for Mg·ATP. In this state, the central β-sheet is twisted ~10° beyond the maximal amount observed in other kinesins. This configuration has only been seen in the nucleotide-free states of myosins-known as the "rigor-like" state. Fitting of this atomic model to electron density maps from cryo-electron microscopy indicates a distinct binding configuration of the motor domain to microtubules. We postulate that these properties of KIF14 are well suited for stabilizing midbody microtubules during cytokinesis., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The kinesin-13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference-induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin-13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin-13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM's role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM-microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.

We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The method involves quantitative comparison of proteolytic peptides from native versus completely dephosphorylated proteins. Dephosphorylation of serine, threonine, and tyrosine residues is achieved by in-gel treatment of the separated proteins with hydrogen fluoride (HF). This chemical dephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreated samples are used to identify phosphopeptides, which can be confirmed and further studied by tandem mass spectrometry (MS/MS). We have applied this method to identify eight known phosphorylation sites of Xenopus Aurora A kinase, as well as several novel sites in the Xenopus chromosome passenger complex (CPC).

While the metaphase spindle maintains a constant shape and size during cell division, its major component microtubules are continuously being polymerized, depolymerized and transported towards the two spindle poles in a process called microtubule poleward flux. This process has been observed in all metazoan cells. Recent studies have indicated that Kinesin-5s, which can drive the relative sliding of microtubules, and kinesin-13s, which regulate microtubule polymerization, are directly involved in microtubule poleward flux. The availability of molecular and chemical tools to perturb protein functions together with improvements in imaging and analytical methods have allowed the examination of these two kinesins' roles in poleward flux at high temporal and spatial resolution. These advances have shed some light on the molecular mechanisms that drive microtubule poleward flux.

Cell-permeable small molecules that inhibit their targets on fast timescales are powerful probes of cell-division mechanisms. Such inhibitors have been identified using phenotype-based screens with chemical libraries. However, the characteristics of compound libraries needed to effectively span cell-division phenotype space, to find probes that target different mechanisms, are not known. Here we show that a small collection of 100 diaminopyrimidines (DAPs) yields a range of cell-division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles are induced by four inhibitors, including one that targets Polo-like kinases (Plks), evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, we found that Plk activity is needed for the assembly and maintenance of bipolar mitotic spindles. Plk inhibition destabilizes kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Further testing of compounds based on 'privileged scaffolds', such as the DAP scaffold, could lead to new cell-division probes and antimitotic agents.

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division, represent alternatives to antimitotic agents that target tubulin. While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol, act through poorly understood allosteric mechanisms, not competing with ATP binding. Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays. We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separation rates in vivo. Furthermore, we found that Eg5 can tether microtubule plus-ends, suggesting an additional microtubule-binding mode for Eg5. Our results demonstrate how members of the kinesin-5 family are likely to function in mitosis, pushing apart interpolar microtubules as well as recruiting microtubules into bundles that are subsequently polarized by relative sliding.

During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.

A full account of the total syntheses of proteasome inhibitors TMC-95A and -B is provided. A key feature of the syntheses involved installation of a cis-propenylamide moiety by a thermal rearrangement of an alpha-silylallyl amide. The scope and mechanism of the enamide-forming reaction are discussed. Also provided are some preliminary results from SAR studies. It was found that simplified analogues can retain the full potency of proteasome inhibition.

Background: Intraflagellar transport (IFT) uses kinesin II to carry a multiprotein particle to the tips of eukaryotic cilia and flagella and a nonaxonemal dynein to return it to the cell body. IFT particle proteins and motors are conserved in ciliated eukaryotes, and IFT-deficient mutants in algae, nematodes, and mammals fail to extend or maintain cilia and flagella, including sensory cilia. In Drosophila, the only ciliated cells are sensory neurons and sperm. no mechanoreceptor potential (nomp) mutations have been isolated that affect the differentiation and function of ciliated sense organs. The nompB gene is here shown to encode an IFT protein. Its mutant phenotypes reveal the consequences of an IFT defect in an insect., Results: Mechanosensory and olfactory neurons in nompB mutants have missing or defective cilia. nompB encodes the Drosophila homolog of the IFT complex B protein IFT88/Polaris/OSM-5. nompB is expressed in the ciliated sensory neurons, and a functional, tagged NOMPB protein is located in sensory cilia and around basal bodies. Surprisingly, nompB mutant males produce normally elongated, motile sperm. Neuronally restricted expression and male germline mosaic experiments show that nompB-deficient sperm are fully functional in transfer, competition, and fertilization., Conclusions: NOMPB, the Drosophila homolog of IFT88, is required for the assembly of sensory cilia but not for the extension or function of the sperm flagellum. Assembly of this extremely long axoneme is therefore independent of IFT.

The proteasome regulates diverse intracellular processes, including cell-cycle progression, antigen presentation, and inflammatory response. Selective inhibitors of the proteasome have great therapeutic potential for the treatment of cancer and inflammatory disorders. Natural cyclic peptides TMC-95A and B represent a new class of noncovalent, selective proteasome inhibitors. To explore the structure-activity relationship of this class of proteasome inhibitors, a series of TMC-95A/B analogues were prepared and analyzed. We found that the unique enamide functionality at the C8 position of TMC-95s can be replaced with a simple allylamide. The asymmetric center at C36 that distinguishes TMC-95A from TMC-95B but which necessitates a complicated separation of the two compounds can be eliminated. Therefore, these findings could lead to the development of more accessible simple analogues as potential therapeutic agents.

p34, a specific p-nitrophenyl phosphatase (pNPPase) was identified and purified from the murine cell line EL4 in a screen for the intracellular molecular targets of the antiinflammatory natural product parthenolide. A BLAST search analysis revealed that it has a high degree of sequence similarity to two yeast alkaline phosphatases. We have cloned, sequenced, and expressed p34 as a GST-tagged fusion protein in Escherichia coli and an EE-epitope-tagged fusion protein in mammalian cells. Using p-nitrophenyl phosphate (pNPP) as a substrate, p34 is optimally active at pH 7.6 with a K(m) of 1.36 mM and K(cat) of 0.052 min(-1). Addition of 1 mM Mg(2+) to the reaction mixture increases its activity by 14-fold. Other divalent metal ions such as Co(2+) and Mn(2+) also stimulated the activity of the enzyme, while Zn(2+), Fe(2+), and Cu(2+) had no effect. Furthermore, both NaCl and KCl enhanced the activity of the enzyme, having maximal effect at 50 and 75 mM, respectively. The enzyme is inhibited by sodium orthovanadate but not by sodium fluoride or okadaic acid. Mutational analysis data suggest that p34 belongs to the group of phosphatases characterized by the sequence motif DXDX(T/V).

Background: Biologically active natural products continue to be useful in the exploration and control of intracellular signaling processes. For example, the sesquiterpene lactone parthenolide from the anti-inflammatory medicinal herb Feverfew (Tanacetum parthenium) appears to inhibit the pro-inflammatory signaling pathway. Parthenolide's direct molecular target, however, remains unknown. We set out to identify the molecular mechanisms of parthenolide's anti-inflammatory activity., Results: A parthenolide affinity reagent was synthesized and shown to bind directly to and inhibit IkappaB kinase beta (IKKbeta), the kinase subunit known to play a critical role in cytokine-mediated signaling. Mutation of cysteine 179 in the activation loop of IKKbeta abolished sensitivity towards parthenolide. Moreover, we showed that parthenolide's in vitro and in vivo anti-inflammatory activity is mediated through the alpha-methylene gamma-lactone moiety shared by other sesquiterpene lactones., Conclusions: In recent years, the multi-subunit IKK complex has been shown to be responsible for cytokine-mediated stimulation of genes involved in inflammation and as such represents an attractive target for pharmaceutical intervention. Our finding that parthenolide targets this kinase complex provides a possible molecular basis for the anti-inflammatory properties of parthenolide. In addition, these results may be useful in the development of additional anti-inflammatory agents.

The proteasome regulates cellular processes as diverse as cell cycle progression and NF-kappaB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 microM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-kappaB activation in vitro and potently blocks in vivo inflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems.

Epoxomicin (1), a peptide alpha',beta'-epoxyketone isolated from the actinomycete strain No.Q996-17, possesses potent in vivo anti-tumor and anti-inflammatory activities. In this paper, we report the first syntheses of epoxomicin, [3H]-epoxomicin, and a biotinylated epoxomicin analog as well as the absolute configuration of the epoxide stereocenter. The natural product and derivatives have permitted the first identification of the proteasome as the specific cellular target of epoxomicin.

Cell cycle progression requires the proteasome-mediated degradation of key regulatory proteins such as cyclins, cyclin-dependent kinase inhibitors, and anaphase-inhibitory proteins. Given the central role of the proteasome in the destruction of these proteins, proteasome inhibition has been proposed as a possible cancer therapy. We report here that dihydroeponemycin, an analogue of the antitumor and antiangiogenic natural product eponemycin, selectively targets the 20S proteasome. Dihydroeponemycin covalently modifies a subset of catalytic proteasomal subunits, binding preferentially to the IFN-gamma-inducible subunits LMP2 and LMP7. Moreover, the three major peptidolytic activities of the proteasome are inhibited by dihydroeponemycin at different rates. In addition, dihydroeponemycin-mediated proteasome inhibition induces a spindle-like cellular morphological change and apoptosis. These results validate the proteasome as a target for antitumor pharmacological intervention and are relevant for the design of novel chemotherapeutic strategies.