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Salt stress is one of the major impairments to agricultural soil that significantly reduces growth and productivity in rice (Oryza sativa L.) and other crop plants. The proteomic mechanisms underlying salt stress tolerance in rice have not been well established. Therefore, a comprehensive understanding of molecular mechanisms associated with salt signaling, salt-toxicity detoxification, and other metabolic mechanisms is essential for elucidating salt tolerance mechanisms in rice as well as ensuring global food security. Recent proteome studies have provided a global proteomic signature of rice cultivars. Integrative studies of proteomic, physiological, and molecular responses under salt stress have provided detailed mechanisms associated with salt stress tolerance in rice. This review explores the proteomic mechanisms with finely-tuned salt-responsive networks in this cereal. Several proteomic processes, including salt sensing and signaling, scavenging of reactive oxygen species (ROS) and stress defense, salt compartmentalization and homeostasis, alterations of cell wall components, modulation of the cytoskeleton, regulation of salt-responsive genes, transcription factors, and protein synthesis, protein folding and processing, protein degradation, and strategies of carbohydrate and energy metabolism for organ development, elucidate extensive molecular mechanisms linked to salt stress responses and tolerance in rice. Further, it is updated the prospects of salt stress tolerance in rice using multi-omics and CRISPR/Cas approaches. These finely-tuned molecular insights will be beneficial to rice breeders and farmers for developing high-yielding, salt-tolerant rice cultivars to achieve global food security.

Parkinson’s disease (PD) is a common and incurable neurodegenerative disorder that arises from the loss of dopaminergic neurons in the substantia nigra and is mainly characterized by progressive loss of motor function. Monogenic familial PD is associated with highly penetrant variants in specific genes, notably the PRKN gene, where homozygous or compound heterozygous loss-of-function variants predominate. PRKN encodes Parkin, an E3 ubiquitin-protein ligase important for protein ubiquitination and mitophagy of damaged mitochondria. Accordingly, Parkin plays a central role in mitochondrial quality control but is itself also subject to a strict protein quality control system that rapidly eliminates certain disease-linked Parkin variants. Here, we summarize the cellular and molecular functions of Parkin, highlighting the various mechanisms by which PRKN gene variants result in loss-of-function. We emphasize the importance of high-throughput assays and computational tools for the clinical classification of PRKN gene variants and how detailed insights into the pathogenic mechanisms of PRKN gene variants may impact the development of personalized therapeutics., Parkinson’s disease (PD) is a common and incurable neurodegenerative disorder that arises from the loss of dopaminergic neurons in the substantia nigra and is mainly characterized by progressive loss of motor function. Monogenic familial PD is associated with highly penetrant variants in specific genes, notably the PRKN gene, where homozygous or compound heterozygous loss-of-function variants predominate. PRKN encodes Parkin, an E3 ubiquitin-protein ligase important for protein ubiquitination and mitophagy of damaged mitochondria. Accordingly, Parkin plays a central role in mitochondrial quality control but is itself also subject to a strict protein quality control system that rapidly eliminates certain disease-linked Parkin variants. Here, we summarize the cellular and molecular functions of Parkin, highlighting the various mechanisms by which PRKN gene variants result in loss-of-function. We emphasize the importance of high-throughput assays and computational tools for the clinical classification of PRKN gene variants and how detailed insights into the pathogenic mechanisms of PRKN gene variants may impact the development of personalized therapeutics.

Canavan disease is an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. The disease is caused by a deficiency of the cytosolic aspartoacylase (ASPA) enzyme, which catalyzes the hydrolysis of N-acetyl-aspartate (NAA), an abundant brain metabolite, into aspartate and acetate. On the physiological level, the mechanism of pathogenicity remains somewhat obscure, with multiple, not mutually exclusive, suggested hypotheses. At the molecular level, recent studies have shown that most disease linked ASPA gene variants lead to a structural destabilization and subsequent proteasomal degradation of the ASPA protein variants, and accordingly Canavan disease should in general be considered a protein misfolding disorder. Here, we comprehensively summarize the molecular and cell biology of ASPA, with a particular focus on disease-linked gene variants and the pathophysiology of Canavan disease. We highlight the importance of high-throughput technologies and computational prediction tools for making genotype–phenotype predictions as we await the results of ongoing trials with gene therapy for Canavan disease., Canavan disease is an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. The disease is caused by a deficiency of the cytosolic aspartoacylase (ASPA) enzyme, which catalyzes the hydrolysis of N-acetyl-aspartate (NAA), an abundant brain metabolite, into aspartate and acetate. On the physiological level, the mechanism of pathogenicity remains somewhat obscure, with multiple, not mutually exclusive, suggested hypotheses. At the molecular level, recent studies have shown that most disease linked ASPA gene variants lead to a structural destabilization and subsequent proteasomal degradation of the ASPA protein variants, and accordingly Canavan disease should in general be considered a protein misfolding disorder. Here, we comprehensively summarize the molecular and cell biology of ASPA, with a particular focus on disease-linked gene variants and the pathophysiology of Canavan disease. We highlight the importance of high-throughput technologies and computational prediction tools for making genotype–phenotype predictions as we await the results of ongoing trials with gene therapy for Canavan disease.

Parkinson’s disease (PD) is a common and incurable neurodegenerative disorder that arises from the loss of dopaminergic neurons in the substantia nigra and is mainly characterized by progressive loss of motor function. Monogenic familial PD is associated with highly penetrant variants in specific genes, notably the PRKN gene, where homozygous or compound heterozygous loss-of-function variants predominate. PRKN encodes Parkin, an E3 ubiquitin-protein ligase important for protein ubiquitination and mitophagy of damaged mitochondria. Accordingly, Parkin plays a central role in mitochondrial quality control but is itself also subject to a strict protein quality control system that rapidly eliminates certain disease-linked Parkin variants. Here, we summarize the cellular and molecular functions of Parkin, highlighting the various mechanisms by which PRKN gene variants result in loss-of-function. We emphasize the importance of high-throughput assays and computational tools for the clinical classification of PRKN gene variants and how detailed insights into the pathogenic mechanisms of PRKN gene variants may impact the development of personalized therapeutics., Parkinson’s disease (PD) is a common and incurable neurodegenerative disorder that arises from the loss of dopaminergic neurons in the substantia nigra and is mainly characterized by progressive loss of motor function. Monogenic familial PD is associated with highly penetrant variants in specific genes, notably the PRKN gene, where homozygous or compound heterozygous loss-of-function variants predominate. PRKN encodes Parkin, an E3 ubiquitin-protein ligase important for protein ubiquitination and mitophagy of damaged mitochondria. Accordingly, Parkin plays a central role in mitochondrial quality control but is itself also subject to a strict protein quality control system that rapidly eliminates certain disease-linked Parkin variants. Here, we summarize the cellular and molecular functions of Parkin, highlighting the various mechanisms by which PRKN gene variants result in loss-of-function. We emphasize the importance of high-throughput assays and computational tools for the clinical classification of PRKN gene variants and how detailed insights into the pathogenic mechanisms of PRKN gene variants may impact the development of personalized therapeutics.

Canavan disease is an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. The disease is caused by a deficiency of the cytosolic aspartoacylase (ASPA) enzyme, which catalyzes the hydrolysis of N-acetyl-aspartate (NAA), an abundant brain metabolite, into aspartate and acetate. On the physiological level, the mechanism of pathogenicity remains somewhat obscure, with multiple, not mutually exclusive, suggested hypotheses. At the molecular level, recent studies have shown that most disease linked ASPA gene variants lead to a structural destabilization and subsequent proteasomal degradation of the ASPA protein variants, and accordingly Canavan disease should in general be considered a protein misfolding disorder. Here, we comprehensively summarize the molecular and cell biology of ASPA, with a particular focus on disease-linked gene variants and the pathophysiology of Canavan disease. We highlight the importance of high-throughput technologies and computational prediction tools for making genotype–phenotype predictions as we await the results of ongoing trials with gene therapy for Canavan disease., Canavan disease is an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. The disease is caused by a deficiency of the cytosolic aspartoacylase (ASPA) enzyme, which catalyzes the hydrolysis of N-acetyl-aspartate (NAA), an abundant brain metabolite, into aspartate and acetate. On the physiological level, the mechanism of pathogenicity remains somewhat obscure, with multiple, not mutually exclusive, suggested hypotheses. At the molecular level, recent studies have shown that most disease linked ASPA gene variants lead to a structural destabilization and subsequent proteasomal degradation of the ASPA protein variants, and accordingly Canavan disease should in general be considered a protein misfolding disorder. Here, we comprehensively summarize the molecular and cell biology of ASPA, with a particular focus on disease-linked gene variants and the pathophysiology of Canavan disease. We highlight the importance of high-throughput technologies and computational prediction tools for making genotype–phenotype predictions as we await the results of ongoing trials with gene therapy for Canavan disease.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5s AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin–proteasome system. This is achieved via recognition of so-called degradation motifs—degrons—that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin–proteasome system. This is achieved via recognition of so-called degradation motifs—degrons—that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.

The analysis and characterization of aging and archaeological proteins are among the latest challenges in analytical chemistry. In the first part of this dissertation thesis, aging proteins were studied using LC-MS. The effects of aging on protein sequences, including amino acid racemization, post-translational modifications, and protein degradation, were studied. Subsequently, a chiral separation method was developer to determine the amino acid enantiomer rates. In the second part of this dissertation thesis, archaeological proteins were studied using nanoLC-MS. Proteomics, called paleoproteomics in this case, was developed as an alternative method for osteoarchaeology and genomics. Based on two sex-dependent forms of amelogenin protein preserved in teeth, both biological sexes were distinguished by nanoLC-MS because of differences in their protein sequences. The developed proteomic approach was designed to be minimally-invasive. This was confirmed by scanning the teeth before and after amelogenin extraction using both scanning electron microscope and micro-computer tomography., Analýza a charakterizace stárnutí a archeologických proteinů patří mezi nejnovější výzvy v analytické chemii. V první části této disertační práce byly studovány stárnoucí proteiny pomocí LC-MS. Byly studovány účinky stárnutí na proteinové sekvence, včetně racemizace aminokyselin, posttranslačních modifikací a degradace proteinů. Byla vyvinuta metoda chirální separace pro stanovení poměrů aminokyselinových enantiomerů. V druhé části práce byly archeologické proteiny studovány pomocí nanoLC-MS. Proteomika, v tomto případě zvaná paleoproteomika, byla vyvinuta jako alternativní metoda k osteoarcheologii a genomice. Na základě dvou forem proteinu amelogenin, který je pohlavně rozdílný, konzerovovaných v zubech, byly obě pohlaví rozlišeny metodou nanoLC-MS na základě rozdílů v sekvencích. Vyvinutý proteomický přístup byl navržen tak, aby byl minimálně invazivní. To bylo potvrzeno skenováním zubů před a po extrakci amelogeninu., Fakulta chemicko-technologická, Dokončená práce s úspěšnou obhajobou

This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. This seminar provides a deeper understanding of the post-translational mechanisms evolved by eukaryotic cells to target proteins for degradation. Students learn how proteins are recognized and degraded by specific machinery (the proteasome) through their previous tagging with another small protein, ubiquitin. Additional topics include principles of ubiquitin-proteasome function, its control of the most important cellular pathways, and the implication of this system in different human diseases. Finally, speculation on the novel techniques that arose from an increased knowledge of the ubiquitin-proteosome system and current applications in the design of new pharmacological agents to battle disease is also covered.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Rett Syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants in the MECP2 gene. While the majority of RTT-causing variants are clustered in the methyl-CpG binding domain and NCoR/SMRT interaction domain, we report a female patient with a functionally uncharacterized MECP2 variant in the C-terminal domain, c.1030C>T (R344W). We functionally characterized MECP2-R344W in terms of protein stability, NCoR/SMRT complex interaction, and protein nuclear localization in vitro. MECP2-R344W cells showed an increased protein degradation rate without significant change in NCoR/SMRT complex interaction and nuclear localization pattern, suggesting that enhanced MECP2 degradation is sufficient to cause a Rett Syndrome-like phenotype. This study highlights the pathogenicity of the C-terminal domain in Rett Syndrome, and demonstrates the potential of targeting MECP2 protein stability as a therapeutic approach. © 2023 The Authors

Meaningful mRNA-mRNA co-expression of METTL21C gene and other DEGs according to n-6/n-3 PUFA ratio.

List of genes significantly correlated with ARRDC3 and METTL21C and their related functions.

Differentially expressed miRNAs (DEmiRNAs) according to n-6/n-3 PUFA ratio.

List of Gene Ontology (GO) terms related to DEGs according to n-6/n-3 PUFA ratio.

Phenotypic values of n-6/n-3 PUFA ratio, sex classification, and batch grouping recorded in 20 Iberian x Duroc pigs

Differentially expressed genes (DEGs) according to n-6/n-3 PUFA ratio.

Meaningful mRNA-mRNA co-expression of ARRDC3 gene and other DEGs according to n-6/n-3 PUFA ratio

The endoplasmic reticulum (ER) orchestrates different cellular processes by which proteins are synthesized, correctly folded, modified and ultimately transported to their final destinations. As part of this crucial biosynthetic process, proteins that are not properly folded and consequently detrimental to normal cellular function are constantly generated. A common signature of many neurodegenerative diseases, including Alzheimer's and Parkinson's, is accumulation and deposition of misfolded proteins that arise when the ability of cells to deal with the burden of misfolded proteins is compromised. In this course, we will explore how the ER quality control machinery ensures that only properly assembled proteins exit the ER while distinguishing between nascent proteins en route to their biologically active folded state from those that are terminally misfolded.

The endoplasmic reticulum (ER) orchestrates different cellular processes by which proteins are synthesized, correctly folded, modified and ultimately transported to their final destinations. As part of this crucial biosynthetic process, proteins that are not properly folded and consequently detrimental to normal cellular function are constantly generated. A common signature of many neurodegenerative diseases, including Alzheimer's and Parkinson's, is accumulation and deposition of misfolded proteins that arise when the ability of cells to deal with the burden of misfolded proteins is compromised. In this course, we will explore how the ER quality control machinery ensures that only properly assembled proteins exit the ER while distinguishing between nascent proteins en route to their biologically active folded state from those that are terminally misfolded.

A moonlighting protein is one, which carries out multiple, often wholly unrelated, functions. The RAD23 protein is a fascinating example of this, where the same polypeptide and the embedded domains function independently in both nucleotide excision repair (NER) and protein degradation via the ubiquitin-proteasome system (UPS). Hence, through direct binding to the central NER component XPC, RAD23 stabilizes XPC and contributes to DNA damage recognition. Conversely, RAD23 also interacts directly with the 26S proteasome and ubiquitylated substrates to mediate proteasomal substrate recognition. In this function, RAD23 activates the proteolytic activity of the proteasome and engages specifically in well-characterized degradation pathways through direct interactions with E3 ubiquitin-protein ligases and other UPS components. Here, we summarize the past 40 years of research into the roles of RAD23 in NER and the UPS., A moonlighting protein is one, which carries out multiple, often wholly unrelated, functions. The RAD23 protein is a fascinating example of this, where the same polypeptide and the embedded domains function independently in both nucleotide excision repair (NER) and protein degradation via the ubiquitin-proteasome system (UPS). Hence, through direct binding to the central NER component XPC, RAD23 stabilizes XPC and contributes to DNA damage recognition. Conversely, RAD23 also interacts directly with the 26S proteasome and ubiquitylated substrates to mediate proteasomal substrate recognition. In this function, RAD23 activates the proteolytic activity of the proteasome and engages specifically in well-characterized degradation pathways through direct interactions with E3 ubiquitin-protein ligases and other UPS components. Here, we summarize the past 40 years of research into the roles of RAD23 in NER and the UPS.

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.

Background: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. Methods: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. Results: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream

Background: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. Methods: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. Results: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream

A moonlighting protein is one, which carries out multiple, often wholly unrelated, functions. The RAD23 protein is a fascinating example of this, where the same polypeptide and the embedded domains function independently in both nucleotide excision repair (NER) and protein degradation via the ubiquitin-proteasome system (UPS). Hence, through direct binding to the central NER component XPC, RAD23 stabilizes XPC and contributes to DNA damage recognition. Conversely, RAD23 also interacts directly with the 26S proteasome and ubiquitylated substrates to mediate proteasomal substrate recognition. In this function, RAD23 activates the proteolytic activity of the proteasome and engages specifically in well-characterized degradation pathways through direct interactions with E3 ubiquitin-protein ligases and other UPS components. Here, we summarize the past 40 years of research into the roles of RAD23 in NER and the UPS., A moonlighting protein is one, which carries out multiple, often wholly unrelated, functions. The RAD23 protein is a fascinating example of this, where the same polypeptide and the embedded domains function independently in both nucleotide excision repair (NER) and protein degradation via the ubiquitin-proteasome system (UPS). Hence, through direct binding to the central NER component XPC, RAD23 stabilizes XPC and contributes to DNA damage recognition. Conversely, RAD23 also interacts directly with the 26S proteasome and ubiquitylated substrates to mediate proteasomal substrate recognition. In this function, RAD23 activates the proteolytic activity of the proteasome and engages specifically in well-characterized degradation pathways through direct interactions with E3 ubiquitin-protein ligases and other UPS components. Here, we summarize the past 40 years of research into the roles of RAD23 in NER and the UPS.

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.

Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. To preserve their fitness, HSCs are particularly dependent on maintaining protein homeostasis (proteostasis). However, how HSCs purge misfolded proteins is unknown. Here, we show that in contrast to most cells that primarily utilize the proteasome to degrade misfolded proteins, HSCs preferentially traffic misfolded proteins to aggresomes in a Bag3-dependent manner and depend on aggrephagy, a selective form of autophagy, to maintain proteostasis in vivo. When autophagy is disabled, HSCs compensate by increasing proteasome activity, but proteostasis is ultimately disrupted as protein aggregates accumulate and HSC function is impaired. Bag3-deficiency blunts aggresome formation in HSCs, resulting in protein aggregate accumulation, myeloid-biased differentiation, and diminished self-renewal activity. Furthermore, HSC aging is associated with a severe loss of aggresomes and reduced autophagic flux. Protein degradation pathways are thus specifically configured in young adult HSCs to preserve proteostasis and fitness but become dysregulated during aging.

miR-30a and miR-30e seed binding interrogation on the 3’ UTR of DEGs.

miR-15b seed binding interrogation on the 3’ UTR of DEGs

Predicted porcine mature miRNAs co-expression with DEGs.

miR-7142 seed binding interrogation on the 3’ UTR of DEGs

Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditis elegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID∗ degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

Background: The p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different protein adaptors. We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis. Methods: As cancer cachexia models, we used mice bearing colon adenocarcinoma C26, human renal carcinoma RXF393, or Lewis lung carcinoma, with breast cancer 4T1-injected mice as controls. As ALS models, we employed 129/SvHsd mice carrying the mutation G93A in human SOD1. The expression of p97 and its adaptors was analysed in their muscles by quantitative real-time polymerase chain reaction (qPCR) and western blot. We electroporated plasmids into muscles or treated mice with disulfiram (DSF) to test the effects of inhibiting p97 and nuclear protein localization protein 4 (Nploc4), one of its adaptors, on atrophy. Results: The mRNA levels of p97 were induced by 1.5-fold to 2-fold in tibialis anterior (TA) of all the cachectic models but not in the non-cachectic 4T1 tumour-bearing mice (P ≤ 0.05). Similarly, p97 was high both in mRNA and protein in TA from 17-week-old SOD1G93A mice (P ≤ 0.01). Electroporation of a shRNA for murine p97 into mouse muscle reduced the fibre atrophy caused by C26 (P = 0.0003) or ALS (P ≤ 0.01). When we interrogated a microarray, we had previously generated for the expression of p97 adaptors, we found Derl1, Herpud1, Nploc4, Rnf31, and Hsp90ab1 induced in cachectic TA from C26-mice (Fold change > 1.2, adjusted P ≤ 0.05). By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one also induced at the protein level by 1.5-fold (P ≤ 0.01). Electroporation of a CRISPR/Cas9 vector against Nploc4 into muscle reduced the fibre atrophy caused by C26 (P = 0.01) or ALS (P ≤ 0.0001). Because DSF uncouples p97 from Nplo

Proteins and peptides are widely used for the prevention, management, and cure of various illnesses. This vast therapeutic potential represents a significant portion of the pharmaceutical industry in the form of vaccines, antibodies, enzymes, and other protein-based therapeutics. During the drug development process, these proteins/peptides are affected by various environmental factors such as temperature and humidity, as well as the presence of pharmaceutical excipients. These affect the physical and chemical stability of the proteins, which may compromise the safety, efficacy, and shelf-life of the product. With this, stability testing is performed to provide data on how such factors affect the degradation of proteins. In this study, various pharmaceutical excipients were tested for their effect on the chemical stability of two therapeutic proteins, GA-Z, and somatropin over a 30-day incubation period at 37°C. The effects of sucrose (40-120 mg/mL), polysorbate 80 (0.05-1 mg/mL), and polyethylene glycol 600 (20-40%) on the chemical stability of GA-Z were tested. Moreover, glycerol concentrations from 10% to 50% were tested for their effect on somatropin. The results of HPLC-UV analysis of both protein samples provide information on how much native protein remained throughout the incubation period. Sucrose and polysorbate 80 demonstrated an increase in GA-Z stability as their concentrations were increased. Unlike GA-Z, there was no existing LC-UV method for the analysis of somatropin and its degradation products. With this, method development and optimization were performed using a BioResolve reversed-phase column. This process focused on optimizing the flow rate, gradient profile, injection volume, and column oven temperature to improve chromatographic resolution and analyte sensitivity. With the use of this developed method, it was shown that increased glycerol concentrations improved the stability of the somatropin. In conjunction with the LC-UV analysis, further, Proteins and peptides are biomolecules that have a wide range of functions in the human body. As such, they are widely used in the treatment and management of various illnesses such as cancer, diabetes, and cardiovascular diseases. This increased the demand for protein-based products such as vaccines, enzymes, and many other drugs. Nowadays, concerted efforts lead to many protein-based drugs in the market and a lot of which are undergoing clinical trials for approval. During the production of the drug, the protein may be affected by various factors such as temperature, humidity, pharmaceutical excipients, and many other stressors. These excipients are the other substances aside from the protein present in a formulation and they typically comprise the majority of the final product. Their function is to ensure that the protein retains its potency and stability until its administration. The loss of stability of a protein is sometimes coupled with the formation of degradation products. This may result in a decrease in the efficacy and shelf-life of the drug and increases the risk to consumers, as they may be toxic. In this study, various substances were tested for their effects on the stability of two therapeutic proteins, GA-Z, and somatropin. Stability was measured in terms the of chemical degradation of the proteins. Chemical degradation is characterized by a change in the chemical properties of the protein, which leads to changes in its structure and function. For GA-Z, substances such as sucrose, polysorbate 80, and polyethylene glycol 600 in varying concentrations were tested to see if they have stabilizing properties. On the other hand, glycerol was tested for its effect on the chemical stability of somatropin. In the stability studies, both proteins were stored in an incubator at 37°C, with and without excipients, for up to 30 days. After the incubation period, a technique called liquid chromatography was used to determine the amount of the chemically degraded p

Optimization of the biogas generation process is important to achieve efficient degradation and high methane yield, and to reduce methane emissions from the digestate. In this study, serial digester systems with two or three biogas reactors were compared with a single reactor, with the aim of improving degree of degradation and methane yield from food waste and assessing adaptation of microbial communities to different reactor steps. All systems had the same total organic load (2.4 g VS/(L d)) and hydraulic retention time (55 days). Serial systems increased methane yield by >5% compared with the single reactor, with the majority of the methane being obtained from the first-step reactors. Improved protein degradation was also obtained in serial systems, with >20% lower outgoing protein concentration compared with the single reactor and increasing NH4+-N concentration with every reactor step. This resulted in separation of high ammonia (>384 mg NH3-N/L) levels from the main methane production, reducing the risk of methanogen inhibition. Methanosarcina dominated the methanogenic community in all reactors, but increases in the hydrogenotrophic genera Methanoculleus and Methanobacterium were observed at higher ammonia levels. Potential syntrophic acetate-oxidizing bacteria, such as MBA03 and Dethiobacteraceae, followed the same trend as the hydrogenotrophic methanogens. Phylum Bacteroidota family Paludibacteraceae was highly abundant in the first steps and then decreased abruptly, potentially linked to an observed decrease in degradation in the last-step reactors. Nevertheless, the results indicated a trend of increasing relative abundance of the potentially proteolytic genera Proteiniphilum and Fastidiosipila with successive reactor steps., Funding: Tekniska Verken i Linkoping AB; Biogas Research Center; Linkoping University; Swedish University of Agricultural Sciences; Swedish Energy Agency [35624- 2]

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1–ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.

Optimization of the biogas generation process is important to achieve efficient degradation and high methane yield, and to reduce methane emissions from the digestate. In this study, serial digester systems with two or three biogas reactors were compared with a single reactor, with the aim of improving degree of degradation and methane yield from food waste and assessing adaptation of microbial communities to different reactor steps. All systems had the same total organic load (2.4 g VS/(L d)) and hydraulic retention time (55 days). Serial systems increased methane yield by >5% compared with the single reactor, with the majority of the methane being obtained from the first-step reactors. Improved protein degradation was also obtained in serial systems, with >20% lower outgoing protein concentration compared with the single reactor and increasing NH4+-N concentration with every reactor step. This resulted in separation of high ammonia (>384 mg NH3-N/L) levels from the main methane production, reducing the risk of methanogen inhibition. Methanosarcina dominated the methanogenic community in all reactors, but increases in the hydrogenotrophic genera Methanoculleus and Methanobacterium were observed at higher ammonia levels. Potential syntrophic acetate-oxidizing bacteria, such as MBA03 and Dethiobacteraceae, followed the same trend as the hydrogenotrophic methanogens. Phylum Bacteroidota family Paludibacteraceae was highly abundant in the first steps and then decreased abruptly, potentially linked to an observed decrease in degradation in the last-step reactors. Nevertheless, the results indicated a trend of increasing relative abundance of the potentially proteolytic genera Proteiniphilum and Fastidiosipila with successive reactor steps., Funding: Tekniska Verken i Linkoping AB; Biogas Research Center; Linkoping University; Swedish University of Agricultural Sciences; Swedish Energy Agency [35624- 2]

Optimization of the biogas generation process is important to achieve efficient degradation and high methane yield, and to reduce methane emissions from the digestate. In this study, serial digester systems with two or three biogas reactors were compared with a single reactor, with the aim of improving degree of degradation and methane yield from food waste and assessing adaptation of microbial communities to different reactor steps. All systems had the same total organic load (2.4 g VS/(L d)) and hydraulic retention time (55 days). Serial systems increased methane yield by >5% compared with the single reactor, with the majority of the methane being obtained from the first-step reactors. Improved protein degradation was also obtained in serial systems, with >20% lower outgoing protein concentration compared with the single reactor and increasing NH4+-N concentration with every reactor step. This resulted in separation of high ammonia (>384 mg NH3-N/L) levels from the main methane production, reducing the risk of methanogen inhibition. Methanosarcina dominated the methanogenic community in all reactors, but increases in the hydrogenotrophic genera Methanoculleus and Methanobacterium were observed at higher ammonia levels. Potential syntrophic acetate-oxidizing bacteria, such as MBA03 and Dethiobacteraceae, followed the same trend as the hydrogenotrophic methanogens. Phylum Bacteroidota family Paludibacteraceae was highly abundant in the first steps and then decreased abruptly, potentially linked to an observed decrease in degradation in the last-step reactors. Nevertheless, the results indicated a trend of increasing relative abundance of the potentially proteolytic genera Proteiniphilum and Fastidiosipila with successive reactor steps., Funding: Tekniska Verken i Linkoping AB; Biogas Research Center; Linkoping University; Swedish University of Agricultural Sciences; Swedish Energy Agency [35624- 2]

Chloroplasts are ancient organelles responsible for photosynthesis and various biosynthetic functions essential to most life on Earth. Many of these functions require tightly controlled regulatory processes to maintain homeostasis at the protein level. One such regulatory mechanism is the ubiquitin-proteasome system whose fundamental role is increasingly emerging in chloroplasts. In particular, the role of E3 ubiquitin ligases as determinants in the ubiquitination and degradation of specific intra-chloroplast proteins. Here, we highlight recent advances in understanding the roles of plant E3 ubiquitin ligases SP1, COP1, PUB4, CHIP, and TT3.1 as well as the ubiquitin-dependent segregase CDC48 in chloroplast function.

Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditiselegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

Tau pathology of the noradrenergic locus coeruleus (LC) is a hallmark of several age-related neurodegenerative disorders, including Alzheimer’s disease. However, a comprehensive neuropathological examination of the LC is difficult due to its small size and rod-like shape. To investigate the LC cytoarchitecture and tau cytoskeletal pathology in relation to possible propagation patterns of disease-associated tau in an unprecedented large-scale three-dimensional view, we utilized volume immunostaining and optical clearing technology combined with light sheet fluorescence microscopy. We examined AT8+ pathological tau in the LC/pericoerulear region of 20 brains from Braak neurofibrillary tangle (NFT) stage 0–6. We demonstrate an intriguing morphological complexity and heterogeneity of AT8+ cellular structures in the LC, representing various intracellular stages of NFT maturation and their diverse transition forms. We describe novel morphologies of neuronal tau pathology such as AT8+ cells with fine filamentous somatic protrusions or with disintegrating soma. We show that gradual dendritic atrophy is the first morphological sign of the degeneration of tangle-bearing neurons, even preceding axonal lesions. Interestingly, irrespective of the Braak NFT stage, tau pathology is more advanced in the dorsal LC that preferentially projects to vulnerable forebrain regions in Alzheimer’s disease, like the hippocampus or neocortical areas, compared to the ventral LC projecting to the cerebellum and medulla. Moreover, already in the precortical Braak 0 stage, 3D analysis reveals clustering tendency and dendro-dendritic close appositions of AT8+ LC neurons, AT8+ long axons of NFT-bearing cells that join the ascending dorsal noradrenergic bundle after leaving the LC, as well as AT8+ processes of NFT-bearing LC neurons that target the 4th ventricle wall. Our study suggests that the unique cytoarchitecture, comprised of a densely packed and dendritically extensively interconnected neuron, QC 20230523

Optimization of the biogas generation process is important to achieve efficient degradation and high methane yield, and to reduce methane emissions from the digestate. In this study, serial digester systems with two or three biogas reactors were compared with a single reactor, with the aim of improving degree of degradation and methane yield from food waste and assessing adaptation of microbial communities to different reactor steps. All systems had the same total organic load (2.4 g VS/(L d)) and hydraulic retention time (55 days). Serial systems increased methane yield by >5% compared with the single reactor, with the majority of the methane being obtained from the first-step reactors. Improved protein degradation was also obtained in serial systems, with >20% lower outgoing protein concentration compared with the single reactor and increasing NH4+-N concentration with every reactor step. This resulted in separation of high ammonia (>384 mg NH3-N/L) levels from the main methane production, reducing the risk of methanogen inhibition. Methanosarcina dominated the methanogenic community in all reactors, but increases in the hydrogenotrophic genera Methanoculleus and Methanobacterium were observed at higher ammonia levels. Potential syntrophic acetate-oxidizing bacteria, such as MBA03 and Dethiobacteraceae, followed the same trend as the hydrogenotrophic methanogens. Phylum Bacteroidota family Paludibacteraceae was highly abundant in the first steps and then decreased abruptly, potentially linked to an observed decrease in degradation in the last-step reactors. Nevertheless, the results indicated a trend of increasing relative abundance of the potentially proteolytic genera Proteiniphilum and Fastidiosipila with successive reactor steps., Funding: Tekniska Verken i Linkoping AB; Biogas Research Center; Linkoping University; Swedish University of Agricultural Sciences; Swedish Energy Agency [35624- 2]

Background: Fungi use light as an environmental signal to regulate developmental transitions that are key aspects of their biological cycles and that are also relevant for their dispersal and infectivity as plant or animal pathogens. In addition, light regulates the accumulation of photoprotective pigments, like carotenoids, and other secondary metabolites. Most fungal light responses occur after changes in gene transcription and we describe here a novel efect of light in the regulation of degradation of VE-1, a key component of the velvet complex, in the model fungus Neurospora crassa. The velvet complex is a fungal-specifc protein complex that coordinates fungal development, secondary metabolism, and light regulation by interacting with other regulators and photoreceptors and modifying gene expression. Results: We have characterized the role of VE-1 during conidiation in N. crassa. In vegetative mycelia, VE-1 is localized in the cytoplasm and nuclei and is required for light-dependent transcription but does not interact with the pho‑ toreceptor and transcription factor WC-1. VE-1 is more stable in light than in darkness during asexual development (conidiation). We have shown that this light efect requires the blue-light photoreceptor WC-1. We have characterized the role of the proteasome, the COP9 signalosome (CSN), and the adaptor component of cullin-RING ubiquitin ligases, FWD-1, in the degradation of VE-1. Conclusions: We propose that this new efect of light allows the fungal cell to adapt quickly to changes in light exposure by promoting the accumulation of VE-1 for the regulation of genes that participate in the biosynthesis of photoprotective pigments.

Background: Fungi use light as an environmental signal to regulate developmental transitions that are key aspects of their biological cycles and that are also relevant for their dispersal and infectivity as plant or animal pathogens. In addition, light regulates the accumulation of photoprotective pigments, like carotenoids, and other secondary metabolites. Most fungal light responses occur after changes in gene transcription and we describe here a novel efect of light in the regulation of degradation of VE-1, a key component of the velvet complex, in the model fungus Neurospora crassa. The velvet complex is a fungal-specifc protein complex that coordinates fungal development, secondary metabolism, and light regulation by interacting with other regulators and photoreceptors and modifying gene expression. Results: We have characterized the role of VE-1 during conidiation in N. crassa. In vegetative mycelia, VE-1 is localized in the cytoplasm and nuclei and is required for light-dependent transcription but does not interact with the pho‑ toreceptor and transcription factor WC-1. VE-1 is more stable in light than in darkness during asexual development (conidiation). We have shown that this light efect requires the blue-light photoreceptor WC-1. We have characterized the role of the proteasome, the COP9 signalosome (CSN), and the adaptor component of cullin-RING ubiquitin ligases, FWD-1, in the degradation of VE-1. Conclusions: We propose that this new efect of light allows the fungal cell to adapt quickly to changes in light exposure by promoting the accumulation of VE-1 for the regulation of genes that participate in the biosynthesis of photoprotective pigments.