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Autophagy-based targeted degradation offers a powerful complement to proteasomal degradation leveraging the capacity and versatility of lysosomes to degrade complex cargo. However, it remains unclear which components of the autophagy-lysosomal pathway are most effective for targeted degradation. Here, we describe two orthogonal induced-proximity strategies to identify autophagy effectors capable of degrading organelles and soluble targets. Recruitment of autophagy cargo receptors, ATG8-like proteins, or the kinases ULK1 and TBK1 is sufficient to trigger mitophagy, while only autophagy cargo receptors capable of self-oligomerization degrade soluble cytosolic proteins. We further report a single-domain antibody against p62 and its use as a heterobifunctional degrader to clear mitochondria. Fusing the p62 single-domain antibody to PINK1 enables selective targeting of damaged mitochondria. Our study highlights the importance of avidity for targeted autophagy and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for organelles or protein aggregates. Using proximity-based screening, protein engineering, and structural analysis, this study describes the development of a p62-based biodegrader for the clearance of organelles and aggregated proteins by autophagy-targeted degradation.

Targeted protein degradation (TPD) is a novel strategy for developing therapeutics against pathogens. Prior to causing malaria, Plasmodium parasites replicate within hepatocytes as liver stages, surrounded by a parasitophorous vacuole membrane (PVM). We hypothesized that TPD can be employed to trigger host-driven degradation of essential liver stage PVM proteins and lead to parasite death. To explore this, we took advantage of the proteolysis-targeting-chimera HaloPROTAC3, a molecule that recruits the host von Hippel-Lindau (VHL) E3 ligase to the HaloTag (HT). Parasites expressing HT fused to the host cytosol-exposed domain of the PVM protein UIS4 (UIS4-HT) were generated in Plasmodium berghei and Plasmodium cynomolgi , but only P. berghei UIS4-HT enabled productive liver stage infection experiments in vitro. Although HaloPROTAC3 triggered the degradation of HT proteins in host cells, it had no impact on the survival of P. berghei UIS4-HT liver stages. Furthermore, HaloPROTAC3 bound to P. berghei UIS4-HT but did not recruit VHL or trigger ubiquitination of the PVM. Overall, although this study did not establish whether host-driven TPD can degrade Plasmodium PVM proteins, it highlights the challenges of developing TPD approaches against novel targets and offers insights for advancing this therapeutic strategy against pathogens.

We generated induced pluripotent stem cells (iPSCs) from patient fibroblasts to yield cell lines containing varying degrees of heteroplasmy for a m.13514 A > G mtDNA point mutation (2 lines) and for a ~6 kb single, large scale mtDNA deletion (3 lines). Long term culture of the iPSCs containing a single, large-scale mtDNA deletion showed consistent increase in mtDNA deletion levels with time. Higher levels of mtDNA heteroplasmy correlated with increased respiratory deficiency. To determine what changes occurred in deletion level during differentiation, teratomas comprising all three embryonic germ layers were generated from low (20%) and intermediate heteroplasmy (55%) mtDNA deletion clones. Regardless of whether iPSCs harbouring low or intermediate mtDNA heteroplasmy were used, the final levels of heteroplasmy in all teratoma germ layers increased to a similar high level (>60%). Thus, during human stem cell division, cells not only tolerate high mtDNA deletion loads but seem to preferentially replicate deleted mtDNA genomes. This has implications for the involvement of mtDNA deletions in both disease and ageing.

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca(2+) levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca(2+) levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington's Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca2+ levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca2+ levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.

Harnessing autophagy for targeted degradation is a promising extension to proteasome-based 18 targeted protein degradation because of the capacity and versatility of lysosomes to degrade large and 19 complex cargo, thus broadening the scope of therapeutic targets. While small-molecule degraders 20 recruiting the autophagy machinery to targets are starting to emerge, it remains unclear which component 21 of the autophagy lysosomal pathway is most efficacious to induce selective target degradation. Here, we 22 describe two orthogonal induced-proximity strategies to identify and prioritize autophagy effectors that are 23 sufficient to degrade organelles as well as soluble targets. We show that induced proximity of different 24 effectors such as autophagy cargo receptors, ATG8-like proteins or the kinases ULK1 and TBK1 are 25 sufficient to trigger mitophagy. In contrast, self-oligomerizing autophagy cargo receptors outperform ATG8-26 like effectors and autophagy-related kinases in clearing a soluble cytosolic protein. We further explore the 27 importance of avidity for targeted degradation via autophagy and reveal that the PB1 domain of p62 fused 28 to a LIR peptide is a minimal degron to induce the degradation of mitochondria as well as cytosolic proteins. 29 By developing a novel and highly selective intrabody against the autophagy cargo receptor p62 into a 30 heterobifunctional degrader, we demonstrate that recruitment of endogenous p62 is sufficient to clear 31 mitochondria. This biodegrader, however, is unable to induce degradation of soluble cytosolic proteins due 32 to its inhibitory effect on p62 self-oligomerization. Our study highlights the importance of avidity and 33 suggests that autophagy cargo receptors are attractive entry points for the development of 34 heterobifunctional degraders for complex targets such as organelles or protein aggregates.Competing Interest StatementZ.T, D.M., C.M., N.S., G.S., L.T., A.P., C.S.M., N.G., M.V., S.S.m A.L., J.-Y.C., L.F., P.H., C.B., F.V., S.G., M.M., F.F., G.M., and B.N. are employees of Novartis Pharma. B.G., L.E., and R.M. are former employees of Novartis.

Background Located in the Eastern Democratic Republic of Congo (South-Kivu), Kalehe and Idjwi are two relatively unexplored territories with little to no research on edible insects even though anthropo-entomophagy practice is widespread. This study therefore aimed at exploring the biodiversity, perception, consumption, availability, host plants, harvesting techniques, and processing techniques of edible insects. Methods Data were collected through a field survey using three techniques, namely structured interviews, direct observations, and insect collection and taxonomy. A total of 260 respondents, 130 in each territory, were interviewed. The field survey focused on inventorying commonly edible insects as well as recording consumer preferences, preference factors, seasonal availability, host plants, harvesting techniques, and processing and preservation methods. Samples for taxonomic characterization were preserved in 70% alcohol. Results Nine edible insects, namely Ruspolia differens Serville 1838, Gryllotalpa Africana Palisot de Beauvois 1805 , Locusta migratoria Linnaeus 1758, Macrotermes subhyalinus Rambur 1842, Gnathocera trivittata Swederus 1787, Rhynchophorus phoenicis Fabricius 1801, Vespula spp. Linnaeus 1758, Apis mellifera Linnaeus 1758, and Imbrasia oyemensis Rougeot 1955, were recorded as being consumed either as larvae, pupae, and adults. Ruspolia differens and M. subhyalinus were reported as the most preferred by consumers in the studied territories. A scatter plot of matrices and Pearson's correlations showed a negative correlation between preference based on taste, size, and shape, as well as perceived nutritional value. Their seasonal availability differs from one species to another and correlated with host plants availability. Harvesting techniques and processing and preservation methods depend on species, local knowledge, and practices. Conclusion The huge edible insect diversity observed in Kalehe and Idjwi is evidence of anthropo-entomophagy practices in the area. In addition to being an important delicacy and traditional foods, edible insects can contribute to food, environmental, and financial security through local business opportunities. Households can rely on edible insects to meet their nutritional needs instead of conventional livestock. Indigenous practices and technologies used for harvesting, processing, and preserving edible insects must be improved to meet international standards to increase the market and capitalize on the economic potential of edible insects.

Background The fall Armyworm (FAW) Spodoptera frugiperda (JE Smith), is currently a devastating pest throughout the world due to its dispersal capacity and voracious feeding behaviour on several crops. A MaxEnt species distributions model (SDM) was developed based on collected FAW occurrence and environmental data’s. Bioclimatic zones were identified and the potential distribution of FAW in South Kivu, eastern DR Congo, was predicted. Results Mean annual temperature (bio1), annual rainfall (bio12), temperature seasonality (bio4) and longest dry season duration (llds) mainly affected the FAW potential distribution. The average area under the curve value of the model was 0.827 demonstrating the model efficient accuracy. According to Jackknife test of variable importance, the annual rainfall was found to correspond to the highest gain when used in isolation. FAWs’ suitable areas where this pest is likely to be present in South Kivu province are divided into two corridors. The Eastern corridor covering the Eastern areas of Kalehe, Kabare, Walungu, Uvira and Fizi territories and the Western corridor covering the Western areas of Kalehe, Kabare, Walungu and Mwenga. Conclusions This research provides important information on the distribution of FAW and bioclimatic zones in South Kivu. Given the rapid spread of the insect and the climatic variability observed in the region that favor its development and dispersal, it would be planned in the future to develop a monitoring system and effective management strategies to limit it spread and crop damage.

A clinical trial byJoseph et al.compared the noise reactivity and tolerance to treatment between a standard and a novel trode designed to reduce the peak focal energy used to deliver the extracorporeal shockwave therapy. With the use of a mobility cart, ~60% of caretakers reported improvement in the quality of life of their dog or cat. A study byChristie et al.evaluated the agreement in body condition and muscle condition when a group of working dogs was evaluated by one handler and three veterinarians.