Biology subjects

Mizrak, A.

Publications and source records attributed to Mizrak, A..

5 recordsLinked to original sources

bioRxiv2026

A membrane homeostatic response to lipid overload coordinates fatty acid metabolism

Excess fatty acids can disrupt membrane and organelle function. Cells buffer fatty acid toxicity by synthesizing and storing triglycerides (TGs) in lipid droplets, but their capacity for TG storage is limited. Here, we impaired TG synthesis in hepatocytes and identified adaptive pathways that restore homeostasis during lipid overload. Transcription responds by activating fatty acid oxidation through peroxisome proliferator-activated receptors, and by suppressing synthesis of new, unsaturated fatty acids through sterol regulatory element-binding protein 1 (SREBP1). Mechanistically, SREBP1 appears to respond to changes in ER membrane fluidity. These findings reveal a homeostatic system monitoring and maintaining the ER membrane, coordinating fatty acid synthesis and oxidation, a finding with broad implications for understanding lipid physiology and developing TG synthesis inhibitors for treating fatty liver disease.

cell biology↗

bioRxiv2024

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways

A hallmark of neurodegenerative diseases is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription, and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

cell biology↗

bioRxiv2024

Single-molecule analysis of protein targeting from the endoplasmic reticulum to lipid droplets

Numerous metabolic enzymes translocate from the ER membrane bilayer to the lipid droplet (LD) monolayer, where they perform essential functions. Mislocalization of certain LD-targeted membrane proteins, including HSD17B13 and PNPLA3, is implicated in metabolic dysfunction-associated steatotic liver disease (MASLD). However, the mechanisms governing the trafficking and accumulation of ER proteins on LDs remain poorly understood. Here, using MINFLUX and HILO single-molecule tracking combined with machine learning, we show that HSD17B13, GPAT4, and the model cargo LiveDrop diffuse at comparable speeds in the ER and on LDs, but become nano-confined upon reaching the LD surface. Mechanistic dissection of LiveDrop targeting revealed that this confinement, along with protein accumulation on LDs, depends on specific residues within its targeting motif. These residues mediate preferential and repeated interactions with nanoscale membrane domains, suggesting that LD-targeted proteins selectively partition into distinct lipid-protein environments that transiently retain and concentrate them at the LD surface. Single-molecule trajectories further revealed bidirectional trafficking of LiveDrop across seipin-containing ER-LD bridges, providing direct evidence for lateral protein transfer across membrane contact sites. These findings establish nanodomain-based confinement as a key mechanism driving selective protein accumulation on LDs and reveal how membrane bridges between organelles facilitate protein sorting.

cell biology↗

bioRxiv2021

Identification of two pathways mediating protein targeting from ER to lipid droplets

Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER. HIGHLIGHTSO_LIProteins localize to LDs either during formation or later through ER-LD bridges C_LIO_LISpecific membrane fusion machinery localizes to LDs and mediates protein targeting C_LIO_LISpecific ER exit site proteins associate with LDs and participate in ERTOLD targeting C_LIO_LIProteomic studies reveal cargoes for early and late ERTOLD targeting C_LI

cell biology↗

bioRxiv2019

Polyanions provide selective control of APC/C interactions with the activator subunit

Transient interactions between the Anaphase-Promoting Complex/Cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitination activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with the ubiquitination of low-affinity substrates. However, engagement with high-affinity substrates blocks the inhibitory effects of polyanions on activator binding and APC/C activity. This mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitination of high-affinity substrates and suppressing ubiquitination of low-affinity substrates.

biochemistry↗