Degradation Of Macromolecules Research Articles

Pulmonary storage.

Interstitial lung diseases (ILDs) are not just a matter of scarring or inflammation in the lung tissue. The lungs can also serve as a repository for products that can be produced in excessive amounts in the human body as a result of disease. Geneticaly based dysfunctions of lysosomal enzymes, which leads to an unefficient degradation and transport of various macromolecules from lysosomes, are considered to be storage diseases sensu stricto. ILDs were described in patients with Gaucher disease, Niemann-Pick disease and Fabry disease. In a broader context, however, the accumulation of various substances in the lung tissue is also encountered in cases of pediatric pulmonary interstitial glycogenosis (PIG), alveolar lipoproteinosis or pulmonary amyloidosis. The cause of PIG is not clear. The disease was first described in 2002 and a lung tissue sample is required to establish this diagnosis. Even though PIG usually goes well in childhood and the patients difficulties spontaneously subside over time, the long-term prognosis of the patients is unknown. Alveolar lipoproteinoses can be acquired (e.g. after massive exposure to silica dust), autoimmune, but also genetically determined. Unlike lysosomal storage diseases, in the case of pulmonary alveolar lipoproteinosis, accumulation of abnormal macromolecules occurs only in the lungs of affected individuals. Similarly, amyloidosis is not a single disease, but a group of diseases with different etiopathogenesis, as a result of which amyloid - a group of different proteins with a distinctvive conformation, which can be deposited in various organs, including the lungs - is formed. The diagnosis of pulmonary alveolarlipoproteinosis is based on the typical appearance and biochemical composition of the fluid obtained by bronchoalveolar lavage, the diagnosis of amyloidosis is histological.

Autophagy modulation in organotypic retinal cultures exposed to high glucose

AbstractPurpose: Diabetic retinopathy (DR) is one of the most serious complications of diabetes mellitus and a leading cause of vision loss among working‐age population in industrialized countries (1, 2). DR has long been considered a microvascular disease (3). However, more recently, neurodegeneration and neuroinflammation emerged as important determinants in the pathogenesis and evolution of the pathology (4). Autophagy, the main catabolic pathway involved in the degradation and recycling of damaged organelles and macromolecules, plays an important role in the pathophysiology of retina neurodegenerative diseases (5). Here we studied the changes occurring in ex‐vivo cultured retinas under normal and high glucose conditions and focused on the autophagy pathway.Methods: Organotypic retinal culture from C57BL/6J mice were exposed to normal (15 mM) (NG) or high (50 mM) glucose (HG) for 7 days. Neuronal survival and glial activation were studied by immunofluorescence with cell subtype specific antibodies. Protein or mRNA levels of autophagy‐related proteins (LC3, p62, Atg5‐12) and inflammatory markers (GFAP, IL‐1β and IL‐6) were studied by western blotting or quantitative PCR.Results: Loss of the neuronal components and reduction of glial reactivity were observed in the retina maintained in NG for 7 days. These cellular changes were only slightly affected by exposure to HG. However, under these conditions an upregulation of inflammatory related proteins and significant changes in the autophagic flux were reported.Conclusions: Our preliminary data suggest that autophagy is modulated in organotypic retinal cultures exposed to HG although further experiments are needed to define its pathological role.References.1. Seewoodhary M. Nurs Stand(2021; 30 36: 71–76.2. Prokofyeva E and Zrenner E. Ophthalmic Res 2012; 7 47: 171–188.3. Cheung CY et al. Diabetologia 2017; 60: 1770–1781.4. Kadłubowska J et al. Curr Neuropharmacol 2016; 14: 831–839.5. Adornetto A et al. Front Cell Dev Biol 2020; 4;8: 121.This work is supported by grants from the Italian Ministry of Education, University and Research: PRIN 2017 protocol number “2017TSHBXZ_002.”

Frequent Occurrence and Metabolic Versatility of Marinifilaceae Bacteria as Key Players in Organic Matter Mineralization in Global Deep Seas.

Transfer of animal and plant detritus of both terrestrial and marine origins to the deep sea occurs on a global scale. Microorganisms play an important role in mineralizing them therein, but these are yet to be identified in situ. To observe key bacteria involved, we conducted long-term in situ incubation and found that members of the family Marinifilaceae (MF) occurred as some of the most predominant bacteria thriving on the new inputs of plant and animal biomasses in the deep sea in both marginal and oceanic areas. This taxon is diverse and ubiquitous in marine environments. A total of 11 MAGs belonging to MF were retrieved from metagenomic data and diverged into four subgroups in the phylogenomic tree. Based on metagenomic and metatranscriptomic analyses, we described the metabolic features and in situ metabolizing activities of different subgroups. The MF-2 subgroup, which dominates plant detritus-enriched cultures, specializes in polysaccharide degradation and lignin oxidation and has high transcriptional activities of related genes in situ. Intriguingly, members of this subgroup encode a nitrogen fixation pathway to compensate for the shortage of nitrogen sources inside the plant detritus. In contrast, other subgroups dominating the animal tissue-supported microbiomes are distinguished from MF-2 with regard to carbon and nitrogen metabolism and exhibit high transcriptional activity for proteolysis in situ. Despite these metabolic divergences of MF lineages, they show high in situ transcriptional activities for organic fermentation and anaerobic respiration (reductions of metal and/or dimethyl sulfoxide). These results highlight the role of previously unrecognized Marinifilaceae bacteria in organic matter mineralization in marine environments by coupling carbon and nitrogen cycling with metal and sulfur. IMPORTANCE Microbial mineralization of organic matter has a significant impact on the global biogeochemical cycle. This report confirms the role of Marinifilaceae in organic degradation in the oceans, with a contribution to ocean carbon cycling that has previously been underestimated. It was the dominant taxon thriving on plant and animal biomasses in our in situ incubator, as well as in whale falls and wood falls. At least 9 subgroups were revealed, and they were widely distributed in oceans globally but predominant in organic-matter-rich environments, with an average relative abundance of 8.3%. Different subgroups display a preference for the degradation of different macromolecules (polysaccharides, lignin, and protein) and adapt to their environments via special metabolic mechanisms.

Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyze lysosomes and early endosomes. Based on the identification of 5376 cross-links, we investigate protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and a heterodimeric structure of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identify >300 putative cargo proteins and confirm eleven substrates for flotillin-dependent endocytosis, including the latrophilin family of adhesion G protein-coupled receptors.

Lysosomal solute and water transport

Lysosomes mediate hydrolase-catalyzed macromolecule degradation to produce building block catabolites for reuse. Lysosome function requires an osmo-sensing machinery that regulates osmolytes (ions and organic solutes) and water flux. During hypoosmotic stress or when undigested materials accumulate, lysosomes become swollen and hypo-functional. As a membranous organelle filled with cargo macromolecules, catabolites, ions, and hydrolases, the lysosome must have mechanisms that regulate its shape and size while coordinating content exchange. In this review, we discussed the mechanisms that regulate lysosomal fusion and fission as well as swelling and condensation, with a focus on solute and water transport mechanisms across lysosomal membranes. Lysosomal H+, Na+, K+, Ca2+, and Cl- channels and transporters sense trafficking and osmotic cues to regulate both solute flux and membrane trafficking. We also provide perspectives on how lysosomes may adjust the volume of themselves, the cytosol, and the cytoplasm through the control of lysosomal solute and water transport.

Combination of long-term 13CO2 labeling and isotopolog profiling allows turnover analysis of photosynthetic pigments in Arabidopsis leaves

BackgroundLiving cells maintain and adjust structural and functional integrity by continual synthesis and degradation of metabolites and macromolecules. The maintenance and adjustment of thylakoid membrane involve turnover of photosynthetic pigments along with subunits of protein complexes. Quantifying their turnover is essential to understand the mechanisms of homeostasis and long-term acclimation of photosynthetic apparatus. Here we report methods combining whole-plant long-term 13CO2 labeling and liquid chromatography - mass spectrometry (LC–MS) analysis to determine the size of non-labeled population (NLP) of carotenoids and chlorophylls (Chl) in leaf pigment extracts of partially 13C-labeled plants.ResultsThe labeling chamber enabled parallel 13CO2 labeling of up to 15 plants of Arabidopsis thaliana with real-time environmental monitoring ([CO2], light intensity, temperature, relative air humidity and pressure) and recording. No significant difference in growth or photosynthetic pigment composition was found in leaves after 7-d exposure to normal CO2 (~ 400 ppm) or 13CO2 in the labeling chamber, or in ambient air outside the labeling chamber (control). Following chromatographic separation of the pigments and mass peak assignment by high-resolution Fourier-transform ion cyclotron resonance MS, mass spectra of photosynthetic pigments were analyzed by triple quadrupole MS to calculate NLP. The size of NLP remaining after the 7-d 13CO2 labeling was ~ 10.3% and ~ 11.5% for all-trans- and 9-cis-β-carotene, ~ 21.9% for lutein, ~ 18.8% for Chl a and 33.6% for Chl b, highlighting non-uniform turnover of these pigments in thylakoids. Comparable results were obtained in all replicate plants of the 13CO2 labeling experiment except for three that were showing anthocyanin accumulation and growth impairment due to insufficient water supply (leading to stomatal closure and less 13C incorporation).ConclusionsOur methods allow 13CO2 labeling and estimation of NLP for photosynthetic pigments with high reproducibility despite potential variations in [13CO2] between the experiments. The results indicate distinct turnover rates of carotenoids and Chls in thylakoid membrane, which can be investigated in the future by time course experiments. Since 13C enrichment can be measured in a range of compounds, long-term 13CO2 labeling chamber, in combination with appropriate MS methods, facilitates turnover analysis of various metabolites and macromolecules in plants on a time scale of hours to days.

THERMAL DECOMPOSITION OF THE PEROXIDE BONDS IN POLYMERS

Peroxide-containing polymers containing diacyl peroxide groups in the main chain and in the side parts of macromolecules were obtained. The thermal decomposition of peroxide groups in macromolecules is well described by the first-order kinetic equation of rate. During the decomposition of O–O groups of polymers containing peroxide groups in the main chain, a decrease in the degree of polymerization of polymers is observed. The solvent in which the process takes place and the nature of the polymer-matrix, which contains peroxide groups, affects the rate of decomposition of peroxide groups and the degree of degradation of polymer macromolecules. Peroxide-containing polymers can be successfully used to obtain block copolymers, crosslinking agents, and macromolecular initiators.

Purification, crystallization and crystallographic analysis of the PorX response regulator associated with the type IX secretion system.

Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported.

Analysis of energy recovery and microbial community in an amalgamated CSTR-UASBs reactor for a three-stage anaerobic fermentation process of cornstalks.

In this study, a continuous stirred-tank reactor (CSTR) coupled with up-flow anaerobic sludge beds (UASBs) reactor was successfully developed for enhancing methane production and carbon recovery rate from cornstalks. Acetic acid production was higher in regions A than in B and C. The methane percentage achieved at 75.98% of total gas and methane production of cornstalks was up to 520.07 mL/g, during the stable operation period. The carbon of recovery rate, represented substrates converted to methane gas, reached 69.32% in stable stage. Microbial community structure analysis revealed that Paludibacter, Prevotella/Clostridium sensu stricto, and Caldisericum were the dominant bacteria for the degradation of cellulose, lignin, and other refractory macromolecules in regions A, B, and C, respectively. Methanobacterium and Methanolinea were the two major genera, accounting for methanogenesis generation.

Controlled alcoholysis of PET to obtain oligomers for the preparation of PET-PLA copolymer

Controlled degradation of macromolecules or natural macromolecules is important for their subsequent utilization. Normally, it is easier to degrade them to monomers than to oligomers in designated molecular weight range, the controlled degradation is much difficult to achieve and has been challenge for the utilization of macromolecules. Here, we proposed a strategy for simultaneous degradation of the inner and outer layers of PET, which avoid the degradation of polymers from the outside to the inside and achieved the controlled degradation. Under high temperature and micro-pressure conditions, PET is transformed from dense to flocculent structure by the swelling effect, which satisfies the need for simultaneous degradation of the inner and outer layers. In addition, a stable “Dawson” type Polyoxometalates (POMs) α2-K8P2W17O61X(H2O)‧16H2O (X = Zn, Mn, Co, Ni, Cu) with limited number of catalytic activity center was synthesized and applied to PET alcoholysis to obtain oligomer with given molecular weight range. Under the optimal conditions, PET: EG: POM catalyst = 1.0: 4.0: 0.02 (wt%), 240 °C, 10 min, PET was completely degraded and obtained the oligomer products (Mn < 2000 g/mol) with 72.1 % yield. By investigating the mechanism of the controlled alcoholysis, the formation of flocculent PET was be the key factor in achieving controlled degradation; hydrogen bonding and coordination of POMs are the key interactions to achieve rapid alcoholysis. Finally, oligomers were applied in the preparation of PET-PLA copolymers to follow sustainable route. The incorporation of bio-based polymers demonstrates better inclusiveness to the environment and contributes to harmonious development.

Exploring effects of MC-LR to lysosome localization by a unique fluorescence-based method

Maintaining lysosomal pH homeostasis is crucial in regulating physiological processes, such as macromolecule degradation, vesicle trafficking, and plasma membrane repair. However, simultaneous investigation of distribution and interrelation of lysosomes and Microcystin-LR (MC-LR) in living systems is restricted by lack of functional molecular tools. To address this critical challenge, herein, we describe a novel probe Bptp-MR can concurrently report MC-LR and lysosomes for the first time. This probe can realize lysosomal localization and imaging before MC-LR enters the cell. However, its fluorescence signals are no longer limited to lysosomes in the presence of MC-LR in cells because of their pH similarity. This result directly indicates that MC-LR interferes with the intracellular acid region in cells. Thus, this work first reported that MC-LR could disrupt the intracellular acidic regions as indicated by its fluorescence interference with lysosomes in cells. The occurrence and development of major diseases related to MC-LR are possibly associated with the interference of MC-LR with the intracellular acid region. The results provide important insights into studying the pathogenesis of major MC-LR-related diseases.

Small molecule carbon source promoting dairy wastewater treatment of Rhodospirillum rubrum by co-metabolism and the establishment of multivariate nonlinear equation.

Rhodospirillum rubrum water treatment technology could recycle bio-resource. However, the inability to degrade macromolecular organics limited its wide application. This paper discussed the feasibility of small molecular carbon source promoting R. rubrum directly treating dairy machining wastewater (DMW) and accumulations for single cell protein and pigment, and establishment of a mathematical model. Six small molecules promoted the degradation of macromolecules (proteins) in DMW. They promoted protease secretion and non-growth matrix (protein) decomposition in DMW through co-metabolism. Among the molecules, 550 mg/L potassium sodium tartrate was the best, protease activity and protein removal rate were increased by 100% compared with control. Then chemical oxygen demand (COD) and protein removal rates reached 80%, the single cell protein, carotenoid and bacterial chlorophyll yields were increased 2 times. Meanwhile, carbon nitrogen ratio (C/N) and food microbial ratio (F/M) were identified as the most important factors by principal component analysis. A multivariate nonlinear equation model between COD removal rate and C/N, F/M, time was established.

Diversity and Ecophysiology of the Genus OLB8 and Other Abundant Uncultured Saprospiraceae Genera in Global Wastewater Treatment Systems.

The Saprospiraceae family within the phylum Bacteroidota is commonly present and highly abundant in wastewater treatment plants (WWTPs) worldwide, but little is known about its role. In this study, we used MiDAS 4 global survey with samples from 30 countries to analyze the abundance and distribution of members of Saprospiraceae. Phylogenomics were used to delineate five new genera from a set of 31 high-quality metagenome-assembled genomes from Danish WWTPs. Newly designed probes for fluorescence in situ hybridization (FISH) revealed rod-shaped morphologies for all genera analyzed, including OLB8, present mostly inside the activated sludge flocs. The genomes revealed potential metabolic capabilities for the degradation of polysaccharides, proteins, and other complex molecules; partial denitrification; and storage of intracellular polymers (glycogen, polyphosphate, and polyhydroxyalkanoates). FISH in combination with Raman microspectroscopy confirmed the presence of intracellular glycogen in Candidatus Brachybacter, Candidatus Parvibacillus calidus (both from the former genus OLB8), and Candidatus Opimibacter, and the presence of polyhydroxyalkanoates in Candidatus Defluviibacterium haderslevense and Candidatus Vicinibacter. These results provide the first overview of the most abundant novel Saprospiraceae genera present in WWTPs across the world and their potential involvement in nutrient removal and the degradation of macromolecules.

High-fidelity imaging of lysosomal enzyme through in situ ordered assembly of small molecular fluorescent probes

As an organelle in cells, lysosomes play an important role in the degradation of biological macromolecules and pathogens. To elucidate the function of lysosomes in normal or disease states, recently, various fluorescent probes have been reported for imaging lysosomal analytes. However, because of the particularity of the lysosomal environment, most of the reported lysosomal fluorescent probes suffered from a series of practical issues such as easy diffusion, low detection signal-to-background ratio and false signal. To address these issues, based on an optimized in situ ordered assembly solid-state fluorophore HDPQ, we herein put forward a new strategy for the design of lysosomal enzymes probes. As a proof concept, we synthesized a fluorescent probe HDPQ-GLU for lysosomal enzyme β-glucuronidase (GLU). Experiment results displayed that compared with general lysosomal probe, the novel lysosomal probe not only exhibited excellent anti-pH interference ability and high signal-to-noise ratio in aqueous solution, but also has excellent long-term in situ imaging ability in the living system. Using this probe, we have realized high-fidelity and long-term in situ tracking GLU in lysosomes of living cells and evaluated the dynamic changes of GLU during the growth period of zebrafish. We anticipate that the new strategy based on the novel in situ ordered assembly solid-state fluorophore HDPQ may be a potential platform for developing fluorescent probes for high-fidelity imaging of lysosomal enzymes.

New Advances in the Regulation of Leaf Senescence by Classical and Peptide Hormones.

Leaf senescence is the last stage of leaf development, manifested by leaf yellowing due to the loss of chlorophyll, along with the degradation of macromolecules and facilitates nutrient translocation from the sink to the source tissues, which is essential for the plants' fitness. Leaf senescence is controlled by a sophisticated genetic network that has been revealed through the study of the molecular mechanisms of hundreds of senescence-associated genes (SAGs), which are involved in multiple layers of regulation. Leaf senescence is primarily regulated by plant age, but also influenced by a variety of factors, including phytohormones and environmental stimuli. Phytohormones, as important signaling molecules in plant, contribute to the onset and progression of leaf senescence. Recently, peptide hormones have been reported to be involved in the regulation of leaf senescence, enriching the significance of signaling molecules in controlling leaf senescence. This review summarizes recent advances in the regulation of leaf senescence by classical and peptide hormones, aiming to better understand the coordinated network of different pathways during leaf senescence.

A Journey on Extracellular Vesicles for Matrix Metalloproteinases: A Mechanistic Perspective.

Matrix metalloproteinases (MMPs) are key players in matrix remodeling and their function has been particularly investigated in cancer biology. Indeed, through extracellular matrix (ECM) degradation and shedding of diverse cell surface macromolecules, they are implicated in different steps of tumor development, from local expansion by growth to tissue invasion and metastasis. Interestingly, MMPs are also components of extracellular vesicles (EVs). EVs are membrane-limited organelles that cells release in their extracellular environment. These “secreted” vesicles are now well accepted players in cell-to-cell communication. EVs have received a lot of interest in recent years as they are also envisioned as sources of biomarkers and as potentially outperforming vehicles for the delivery of therapeutics. Molecular machineries governing EV biogenesis, cargo loading and delivery to recipient cells are complex and still under intense investigation. In this review, we will summarize the state of the art of our knowledge about the molecular mechanisms implicated in MMP trafficking and secretion. We focus on MT1-MMP, a major effector of invasive cell behavior. We will also discuss how this knowledge is of interest for a better understanding of EV-loading of MMPs. Such knowledge might be of use to engineer novel strategies for cancer treatment. A better understanding of these mechanisms could also be used to design more efficient EV-based therapies.

Draft genome of Gongronella butleri reveals the genes contributing to its biodegradation potential

BackgroundGongronella butleri is a fungus with many industrial applications including the composting of solid biowaste. Kerala Agricultural University, India, has developed a microbial consortium of which GbKAU strain of G. butleri is a major component. Even with great industrial significance, genome of this fungus is not published, and the genes and pathways contributing to the applications are not understood. This study had the objective to demonstrate the solid biowaste decomposing capability of the strain, to sequence and annotate the genome, and to reveal the genes and pathways contributing to its biodegradation potential. ResultsStrain GbKAU of G. butleri isolated and purified from the organic compost was found to produce higher levels of laccase and amylase, compared to Bacillus subtilis which is being widely used in biosolid waste management. Both were shown to be equally efficient in the in vivo composting capabilities. Whole genome sequencing has given ~11 million paired-end good quality reads. De novo assembly using dual-fold approach has yielded 44,639 scaffolds with draft genome size of 29.8 Mb. A total of 11,428 genes were predicted and classified into 359 groups involved in diverse pathways, of which 14 belonged to the enzymes involved in the degradation of macromolecules. Seven previously sequenced strains of the fungus were assembled and annotated. A direct comparison showed that the number of genes present in those strains was comparable to our strain, while all the important biodegrading genes were conserved across the genomes. Gene Ontology analysis had classified the genes according to their molecular function, biological process, and cellular component. A total of 104,718 SSRs were mined and classified to mono- to hexa-nucleotide repeats. The variant analysis in comparison with the closely related genus Cunninghamella has revealed 1156 variants. ConclusionsApart from demonstrating the biodegradation capabilities of the GbKAU strain of G. butleri, the genome of this industrially important fungus was sequenced, de novo assembled, and annotated. GO analysis has classified the genes based on their functions, and the genes involved in biodegradation were revealed. Biodegradation potential, genome features in comparison with other strains, and the functions of the identified genes are discussed.

Non-coding RNAs associated with autophagy and their regulatory role in cancer therapeutics.

Cancer widely affects the world's health population and ranks second leading cause of death globally. Because of poor prognosis of various types of cancer such as sarcoma, lymphoma, adenomas etc., their high recurrence and metastasis rate and low early diagnosis rate have become concern lately. Role of autophagy in cancer progression is being studied since long. Autophagy is cell's self-degradative mechanism towards stress and has role in degradation of the cytoplasmic macromolecules which has potential to damage other cytosolic molecules. Autophagy can promote as well as inhibit tumorigenesis depending upon the associated protein combinations in cancer cells. Recent studies have shown that non-coding RNAs (ncRNAs) do not code for protein but play essential role in modulation of gene expression. At transcriptional level, different ncRNAs like lncRNAs, miRNAs and circRNAs directly or indirectly affect different stages of autophagy like autophagy-dependent and non-apoptotic cell death in cancer cells. This review focuses on the involvement of ncRNAs in autophagy and the modulation of several cancer signal transduction pathways in cancers such as lung, breast, prostate, pancreatic, thyroid, and kidney cancer.

Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.

Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

ATG4A regulates human erythroid maturation and mitochondrial clearance

AbstractAutophagy is a self-degradation pathway that is essential for erythropoiesis. During erythroid differentiation, autophagy facilitates the degradation of macromolecules and the programmed clearance of mitochondria. Impaired mitochondrial clearance results in anemia and alters the lifespan of red blood cells in vivo. While several essential autophagy genes contribute to autophagy in erythropoiesis, little is known about erythroid-specific mediators of this pathway. Genetic analysis of primary human erythroid and nonerythroid cells revealed the selective upregulation of the core autophagy gene ATG4A in maturing human erythroid cells. Because the function of ATG4A in erythropoiesis is unknown, we evaluated its role using an ex vivo model of human erythropoiesis. Depletion of ATG4A in primary human hematopoietic stem and progenitor cells selectively impaired erythroid but not myeloid lineage differentiation, resulting in reduced red cell production, delayed terminal differentiation, and impaired enucleation. Loss of ATG4A impaired autophagy and mitochondrial clearance, giving rise to reticulocytes with retained mitochondria and autophagic vesicles. In summary, our study identifies ATG4A as a cell type–specific regulator of autophagy in erythroid development.