Biology subjects

Morano, K. A.

Publications and source records attributed to Morano, K. A..

5 recordsLinked to original sources

bioRxiv2026

Intermolecular disulfide bond formation promotes Hsp42 higher-order assembly and shapes client selection in yeast

Cellular redox homeostasis plays a critical role in regulating protein function, including chaperone activity, through reversible oxidation of cysteine and methionine residues. Previously, we found that budding yeast cells experiencing redox imbalance due to inactivated thioredoxin reductase (trr1{Delta}) activate the heat shock response and induce hyperaccumulation of the small heat shock protein/sequestrase Hsp42 with misfolded proteins. Building on that finding, this study identified cysteine 127 (C127) within Hsp42 as a redox-active residue that becomes oxidized in trr1{Delta} cells, upon treatment with the powerful oxidant hydrogen peroxide, or by exposure to the cysteine crosslinker divinyl sulfone (DVSF). In trr1{Delta} cells, C127 oxidation promoted intermolecular disulfide bond formation and contributed to Hsp42 homo-oligomerization. We show that stable oligomerization requires both the prion-like domain (PrLD) and C127 oxidation. While Hsp42-GFP formed prominent persistent foci in trr1{Delta} cells, replacement of C127 with non-thiol reactive serine decreased foci formation. Furthermore, the C127S mutation diminished Hsp42 oligomerization and sedimentability. Immunoprecipitation coupled with mass spectrometry analysis revealed that Hsp42 in trr1{Delta} cells preferentially associated with mitochondrial precursor proteins accumulated in the cytoplasm, as well as oxidation-reduction enzymes. The observed client selectivity was altered by the C127S mutation that diversified the spectrum of Hsp42-associated proteins. Collectively, these findings identify Cys127 as a redox-active switch that regulates Hsp42 assembly, foci formation, stability, and client specificity in response to oxidative stress.

cell biology↗

bioRxiv2025

Functional Characterization of Hsp110 in Drosophila Reveals its Essential and Dosage-Sensitive Role in Nervous System Integrity

The Hsp70 molecular chaperone system is the front line of defense in maintaining cellular proteostasis. In eukaryotes, ATP/ADP nucleotide exchange in the Hsp70 chaperone cycle is stimulated by Hsp110, a divergent member of the Hsp70 chaperone superfamily and co-chaperone of Hsp70. Hsp110 is also a known modifier of neurodegenerative and other protein misfolding-related disorders. Biochemical aspects of Hsp110 chaperone functions have been characterized in vitro, and pathway interactions have been extensively characterized genetically in yeast model systems; however, a detailed understanding of its physiological roles in metazoans, particularly in the nervous system has not been carried out. Taking advantage of the single Hsp110-encoding gene in the Drosophila genome, we conducted a comprehensive investigation of its expression and function in this animal model. Notably, Drosophila and human Hsp110 share significant similarity in their sequence, structure, and splicing variants. At the protein level, Hsp110 is ubiquitously expressed, with both cytosolic and nuclear distribution in a tissue-dependent manner. Functionally, while Hsp110 is dispensable for cell proliferation in developing larvae, it is essential for long-term cell survival and normal development of the nervous system, including non-autonomous effects on neuronal differentiation and glial cell migration. Furthermore, despite being identified as a potent suppressor of protein aggregation and neurotoxicity in multiple neurodegenerative diseases, higher levels of Hsp110 are detrimental in flies. Overexpression of Hsp40, another key co-chaperone of Hsp70, can mimic this effect. However, simultaneous overexpression of both Hsp40 and Hsp110 does not further exacerbate their detrimental effect. Together, these results demonstrate a critical role of Hsp110 in neuronal development and cell survival, and further suggest that in vivo, the levels and activities of Hsp110 and Hsp40 co-chaperones need to be properly balanced. Furthermore, this work supports the contention that the Hsp70 chaperone network must be considered as a whole when targeted for potential therapeutic purposes to meet the complex pathophysiological demands in multicellular organisms.

developmental biology↗

bioRxiv2023

Cytoplasmic redox imbalance in the thioredoxin system activates Hsf1 and results in hyperaccumulation of the sequestrase Hsp42 with misfolded proteins

Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1{Delta} cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a mechanism of long-term sequestration.

cell biology↗

bioRxiv2022

Redox regulation of yeast Hsp70 modulates protein quality control while directly triggering an Hsf1-dependent cytoprotective response

Neurodegenerative disease affects millions of Americans every year, through diagnoses such as Alzheimers, Parkinsons, and Huntingtons diseases. One factor linked to formation of these aggregates is damage sustained to proteins by oxidative stress. Cellular protein homeostasis (proteostasis) relies on the ubiquitous Hsp70 chaperone family. Hsp70 activity has been previously shown to be modulated by modification of two key cysteines in the ATPase domain by oxidizing or thiol-modifying compounds. To investigate the biological consequences of cysteine modification on the Hsp70 Ssa1 in budding yeast, we generated cysteine null (cysteine to serine) and oxidomimetic (cysteine to aspartic acid) mutant variants of both C264 and C303 and demonstrate reduced ATP binding, hydrolysis and protein folding properties in both the oxidomimetic as well as hydrogen peroxide-treated Ssa1. In contrast, cysteine nullification rendered Ssa1 insensitive to oxidative inhibition. The oxidomimetic ssa1-2CD (C264D, C303D) allele was unable to function as the sole Ssa1 isoform in yeast cells and also exhibited dominant negative effects on cell growth and viability. Ssa1 binds to and represses Hsf1, the major transcription factor controlling the heat shock response, and the oxidomimetic Ssa1 failed to stably interact with Hsf1, resulting in constitutive activation of the heat shock response. Consistent with the in vitro findings, ssa1-2CD cells were compromised for de novo folding, post-stress protein refolding and in regulated degradation of a model terminally misfolded protein. Together these findings pinpoint Hsp70 as a key link between oxidative stress and proteostasis, information critical to understanding cytoprotective systems that prevent and manage cellular insults underlying complex disease states.

cell biology↗

bioRxiv2021

Suppression of aggregate and amyloid formation by a novel intrinsically disordered region in metazoan Hsp110 chaperones

Molecular chaperones maintain protein homeostasis (proteostasis) by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to the onset of numerous neurodegenerative disorders including Alzheimers, Parkinsons, and Huntingtons disease. Hsp110 is related to the canonical Hsp70 class of protein folding molecular chaperones and interacts with Hsp70 as a nucleotide exchange factor (NEF), promoting rapid cycling of ADP for ATP. In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate binding domain (SBD) whose biological roles remain undefined. Previous work in Drosophila melanogaster has shown that loss of the sole Hsp110 gene (Hsc70cb) accelerates the aggregation of polyglutamine (polyQ)-expanded human Huntingtin, while its overexpression protects against polyQ-mediated neuronal cell death. We hypothesize that in addition to its role as an Hsp70 NEF, Drosophila Hsp110 may function in the fly as a protective protein "holdase", preventing the aggregation of unfolded polypeptides via the SBD-{beta} subdomain. Using an in vitro protein aggregation assay we demonstrate for the first time that Drosophila Hsp110 effectively prevents aggregation of the model substrate citrate synthase. We also report the discovery of a redundant and heretofore unknown potent holdase capacity in a 138 amino-acid region of Hsp110 carboxyl-terminal to both SBD-{beta} and SBD- (henceforth called the C-terminal extension). This sequence is highly conserved in metazoan Hsp110 genes, completely absent from fungal representatives, including Saccharomyces cerevisiae SSE1, and is computationally predicted to contain an intrinsically disordered region (IDR). We demonstrate that this IDR sequence within the human Hsp110s, Apg-1 and Hsp105, inhibits the formation of amyloid A{beta}-42 and -synuclein fibrils in vitro but cannot mediate fibril disassembly. Together these findings demonstrate the existence of a second independent, passive holdase property of metazoan Hsp110 chaperones capable of suppressing both general protein aggregation and amyloidogenesis and raise the possibility of exploitation of this IDR for therapeutic benefit in combating neurodegenerative disease.

biochemistry↗