Biology subjects

Klionsky, D. J.

Publications and source records attributed to Klionsky, D. J..

8 recordsLinked to original sources

bioRxiv2026

Genome-wide analysis reveals the importance of histone acetyltransferase Esa1 in transcriptional regulation during nitrogen starvation

Macroautophagy/autophagy is a process that degrades intracellular components and is strongly triggered by nitrogen starvation (-N). Some ATG (autophagy related) genes are activated at the transcriptional level in nitrogen starvation; however, a full understanding of transcriptional induction and the role of chromatin during this process remains unclear. To address this, we measured the occupancy of RNA polymerase II (Pol II), histone H3, and acetylated H4 (H4Ac) under nutrient-rich and -N conditions by ChIP-seq. We found that most genes are rapidly downregulated within 15-30 min, including ribosomal protein (RP) and biogenesis (RiBi) genes. Meanwhile, genes involved in amino acid (AA) biosynthesis are upregulated, along with many ATG genes. Unexpectedly, RP and RiBi genes were reinduced by 3 hours. Furthermore, many upregulated genes remained active during prolonged starvation. Histones are typically removed from promoters during transcription activation. Consistent with this, we found that most induced genes exhibited histone eviction and increased H4 acetylation at their promoters, suggesting a possible role for histone acetylation in their activation. In line with this, depleting Esa1, an essential H4 histone acetyltransferase, nearly abolished the induction of ribosomal biosynthetic genes and many AA biosynthetic genes. Sustained activation of many genes during prolonged starvation highlights the vital role of transcription in supporting autophagy and cell survival. This is the first comprehensive study to detail changes in chromatin, histone acetylation, and transcription during nitrogen starvation, highlighting the importance of Esa1 and H4Ac in this process.

genetics↗

bioRxiv2024

Sex and Region-Specific Disruption of Autophagy and Mitophagy in Alzheimer's Disease: Linking Cellular Dysfunction to Cognitive Decline

Macroautophagy and mitophagy are critical processes in Alzheimers disease (AD), yet their links to behavioral outcomes, particularly sex-specific differences, are not fully understood. This study investigates autophagy (LC3B-II, SQSTM1) and mitophagy (BNIP3L, BNIP3, BCL2L13) markers in the cortex and hippocampus of male and female 3xTg-AD mice, using western blotting, transmission electron microscopy (TEM), and behavioral tests (novel object recognition and novel object placement). Significant sex-specific differences emerged: female 3xTg-AD mice exhibited autophagosome accumulation due to impaired degradation in the cortex, while males showed fewer autophagosomes, especially in the hippocampus, without significant degradation changes. TEM analyses demonstrated variations in mitochondrial and mitophagosome numbers correlated with memory outcomes. Females had enhanced mitophagy, with higher BNIP3L and BCL2L13 levels, whereas males showed elevated BNIP3 dimers. Cognitive deficits in females correlated with mitochondrial dysfunction in the cortex, while in males, higher LC3B-II levels associated positively with cognitive performance, suggesting protective autophagy effects. Using machine learning, we predicted mitophagosome and mitochondrial numbers based on behavioral data, pioneering a predictive approach to cellular outcomes in AD. These findings underscore the importance of sex-specific regulation of autophagy and mitophagy in AD and support personalized therapeutic approaches targeting these pathways. Integrating machine learning emphasizes its potential to advance neurodegenerative research. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/621097v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1e50ceeorg.highwire.dtl.DTLVardef@b41ac0org.highwire.dtl.DTLVardef@4863d3org.highwire.dtl.DTLVardef@91b2d0_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractSex-specific differences in autophagy and mitophagy regulation in Alzheimers disease (AD) are highlighted. Female 3xTg-AD mice show autophagosome accumulation and cognitive deficits, while males exhibit variations in mitophagy markers and behavior.

neuroscience↗

bioRxiv2024

TgATG9 is required for autophagosome biogenesis and maintenance of chronic infection in Toxoplasma gondii

Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cells digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9{Delta} yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.

microbiology↗

bioRxiv2024

Unlocking a New Path: An Autophagometer that Measures Flux Using a Non-Fluorescent Immunohistochemistry Method

Macroautophagy/autophagy, a crucial cellular process, is typically measured using fluorescence-based techniques, which can be costly, complex, and impractical for clinical settings. In this paper, we introduce a novel, cost-effective, non-fluorescent immunohistochemistry (IHC) method for evaluating autophagy flux. This technique, based on antigen-antibody reactions and chromogenic detection, provides clear, quantifiable results under standard light microscopy, eliminating the need for expensive equipment and specialized reagents. Our method simplifies technical requirements, making it accessible to routine clinical laboratories and research settings with limited resources. By comparing our approach with traditional fluorescence methods, we demonstrate its superior effectiveness, cost-efficiency, and applicability to patient samples. This innovative technique has the potential to significantly advance autophagy research and improve clinical diagnostics, offering a practical and robust tool for studying autophagy mechanisms in diseases such as cancer and neurodegenerative disorders. Our non-fluorescent IHC method represents a significant step forward in evaluating autophagy flux, making it more accessible and reliable, with the promise of enhancing our understanding and treatment of autophagy-related diseases.

molecular biology↗

bioRxiv2022

Intracellular level of S. cerevisiae Rad51 is regulated via proteolysis in a SUMO- and Ubiquitin-dependent manner

Among various DNA lesions, the DNA double-strand breaks are particularly deleterious; especially, when an error-free repair pathway is unavailable, and the cell takes the risk of using the error-prone recombination pathways to repair the DNA breaks, resume the cell cycle, and continue growth. The latter comes at the expense of decreased well-being of the cells due to genome rearrangements. One of the major players involved in recombinational repair of DNA damage is Rad51 recombinase, a protein responsible for presynaptic complex formation. We previously noticed that the level of this protein is strongly increased when illegitimate recombination is engaged in repair. The regulation of Rad51 protein turnover is not known; therefore, we decided to look closer at this issue because we expect that an excessively high level of Rad51 may lead to genome instability. Here we show that the level of Rad51 is regulated via the ubiquitin-dependent proteolytic pathway. The ubiquitination of Rad51 depends on multiple E3 enzymes, including SUMO-targeted ubiquitin ligases. We also demonstrate that Rad51 can be modified by both ubiquitin and SUMO. Moreover, these modifications may lead to opposite effects. Ubiquitin-dependent degradation depends on Rad6, Rad18, Slx8, Dia2 and the anaphase-promoting complex. Rsp5-dependent ubiquitination leads to Rad51 stabilization.

genetics↗

bioRxiv2021

The human disease gene CLEC16A encodes an intrinsically disordered protein region required for mitochondrial quality control

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. While CLEC16A has ubiquitin ligase activity, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs pancreatic {beta}-cell mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases its self-ubiquitination and destabilizes CLEC16A, thus impairing formation of a critical CLEC16A-dependent mitophagy complex. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we clarify how an IDPR in CLEC16A prevents diabetes, thus implicating the disruption of IDPRs as novel pathological contributors to diabetes and other CLEC16A-associated diseases.

molecular biology↗

bioRxiv2019

TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy

Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the upregulation of autophagy and vacuole proteins involved in recycling, but also result in the downregulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Downregulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

cell biology↗

bioRxiv2017

The Function of the COPII Gene Paralogs SEC23A and SEC23B Are Interchangeable In Vivo

SEC23 is a core component of the coat protein-complex II (COPII)-coated vesicle, which mediates transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi1-3. Mammals express 2 paralogs for SEC23 (SEC23A and SEC23B). Though the SEC23 gene duplication dates back >500 million years, both SEC23s are ~85% identical at the amino acid sequence level. In humans, deficiency for SEC23A or SEC23B results in cranio-lenticulo-sutural dysplasia4 or congenital dyserythropoietic anemia type II (CDAII), respectively5. The disparate human syndromes and reports of secretory cargos with apparent paralog-specific dependence6,7, suggest unique functions for the two SEC23 paralogs. Here we show indistinguishable intracellular interactomes for human SEC23A and SEC23B, complementation of yeast SEC23 by both human and murine SEC23A/B paralogs, and the rescue of lethality resulting from Sec23b disruption in zebrafish by a Sec23a-expressing transgene. Finally, we demonstrate that the Sec23a coding sequence inserted into the endogenous murine Sec23b locus fully rescues the mortality and severe pancreatic phenotype previously reported with SEC23B-deficiency in the mouse8-10. Taken together, these data indicate that the disparate phenotypes of SEC23A and SEC23B deficiency likely result from evolutionary shifts in gene expression program rather than differences in protein function, a paradigm likely applicable to other sets of paralogous genes. These findings also suggest the potential for increased expression of SEC23A as a novel therapeutic approach to the treatment of CDAII, with potential relevance to other disorders due to mutations in paralogous genes.

evolutionary biology↗