Mechanism Of Protein Degradation Research Articles

Manganese Exposure Enhances the Release of Misfolded α-Synuclein via Exosomes by Impairing Endosomal Trafficking and Protein Degradation Mechanisms

Manganese Exposure Enhances the Release of Misfolded α-Synuclein via Exosomes by Impairing Endosomal Trafficking and Protein Degradation Mechanisms

RNA-Sequencing Identification of Genes Supporting HepG2 as a Model Cell Line for Hepatocellular Carcinoma or Hepatocytes

Background/Objectives: Cell lines do not faithfully replicate the authentic transcriptomic condition of the disease under study. The HepG2 cell line is widely used for studying hepatocellular carcinoma (HCC), but not all biological processes and genes exhibit congruent expression patterns between cell lines and the actual disease. The objective of this study is to perform a comparative transcriptomic analysis of the HepG2 cell line, HCC, and primary hepatocytes (PH) in order to identify genes suitable for research in HepG2 as a model for PH or HCC research. Methods: We conducted a differential expression analysis between publicly available data from HCC patients, PH, and HepG2. We examined specific overlaps of differentially expressed genes (DEGs) in a pairwise manner between groups in order to obtain a valuable gene list for studying HCC or PH using different parameter filtering. We looked into the function and druggability of these genes. Conclusions: In total, we identified 397 genes for HepG2 as a valuable HCC model and 421 genes for HepG2 as a valuable PH model, and with more stringent criteria, we derived a smaller list of 40 and 21 genes, respectively. The majority of genes identified as a valuable set for the HCC model are involved in DNA repair and protein degradation mechanisms. This research aims to provide detailed guidance on gene selection for studying diseases like hepatocellular carcinoma, primary hepatocytes, or others using cell lines.

Characteristics and biological mechanism of protein degradation by the black solider fly (Hermetia illucens L.) larvae gut strain Bacillus subtilis S4

The black solider fly larvae (BSFL) can efficiently convert nitrogen in organic waste into insect protein. Bacillus subtilis S4, an efficient protein-degrading bacterium from the BSFL gut, was isolated and identified to explore the mechanism of nutrient metabolism underlying BSFL nitrogen utilization. Results showed that B. subtilis S4 could effectively increase larval biomass in a bean stem bioconversion system. In vitro high-performance liquid chromatography analysis showed that B. subtilis S4 completely degraded casein into Val, Ile, Phe, Leu, Tyr, Lys, Gly, and Met. Various protease genes with secretion expression ability were annotated in B. subtilis S4. They included peptidoglycan DL - endopeptidase, aminopeptidase, extracellular metalloprotease, peptidoglycan endopeptidase, and peptidase M15. Therefore, the BSFL intestinal microbe B. subtilis S4 could effectively degrade protein and promote larval biomass accumulation, which could provide novel insights into the combined conversion of organic waste into proteins by microbes and insects.

Decreased SREBP2 of the striatal cell relates to disrupted protein degradation in Huntington’s disease

This study delineated the intricate relation between cholesterol metabolism, protein degradation mechanisms, and the pathogenesis of Huntington’s disease (HD). Through investigations using both animal models and cellular systems, we have observed significant alterations in cholesterol levels, particularly in the striatum, which is the primary lesion site in HD. Our findings indicate the dysregulation of cholesterol metabolism-related factors, such as LDLR and SREBP2, in HD, which may contribute to disease progression. Additionally, we uncovered disruptions in protein degradation pathways, including decreased neddylated proteins and dysregulated autophagy, which further exacerbated HD pathology. Moreover, our study highlighted the potential therapeutic implications of targeting these pathways. By restoring cholesterol levels and modulating protein degradation mechanisms, particularly through interventions, such as MLN4924, we observed potential improvements in cellular function, as indicated by the increased BDNF levels. These insights underscore the importance of simultaneously addressing cholesterol metabolism and protein degradation to alleviate HD pathology. Collectively, this study provides a basic understanding of the interplay between the decrease of SREBP2 and the dysfunctional protein degradation system derived from disrupted cholesterol metabolism in HD and HD cells.

Near UV and Visible Light-Induced Degradation of Bovine Serum Albumin and a Monoclonal Antibody Mediated by Citrate Buffer and Fe(III): Reduction vs Oxidation Pathways.

Light exposure during manufacturing, storage, and administration can lead to the photodegradation of therapeutic proteins. This photodegradation can be promoted by pharmaceutical buffers or impurities. Our laboratory has previously demonstrated that citrate-Fe(III) complexes generate the •CO2- radical anion when photoirradiated under near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) [Subelzu, N.; Schöneich, C. Mol. Pharmaceutics 2020, 17 (11), 4163-4179; Zhang, Y. Mol. Pharmaceutics 2022, 19 (11), 4026-4042]. Here, we evaluated the impact of citrate-Fe(III) on the photostability and degradation mechanisms of disulfide-containing proteins (bovine serum albumin (BSA) and NISTmAb) under pharmaceutically relevant conditions. We monitored and localized competitive disulfide reduction and protein oxidation by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis depending on the reaction conditions. These competitive pathways were affected by multiple factors, including light dose, Fe(III) concentration, protein concentration, the presence of oxygen, and light intensity.

All-Trans Retinoic Acid-Induced Cell Surface Heat Shock Protein 90 Mediates Tau Protein Internalization and Degradation in Human Microglia.

This study investigates the role of all-trans retinoic acid (ATRA) in modulating the expression of heat shock protein 90 (Hsp90) and its influence on the uptake and degradation of tau proteins in immortalized human microglia cells. We demonstrate that ATRA significantly upregulates Hsp90 expression in a concentration-dependent manner, enhancing both extracellular and intracellular Hsp90 levels. Our results show that ATRA-treated cells exhibit increased tau protein uptake via caveolae/raft-dependent endocytosis pathways. This uptake is mediated by surface Hsp90, as evidenced by the inhibition of tau internalization using an extracellular Hsp90-selective inhibitor. Further, we establish that the exogenously added full-sized monomeric tau proteins bind to Hsp90. The study also reveals that ATRA-enhanced tau uptake is followed by effective degradation through both lysosomal and proteasomal pathways. We observed a significant reduction in intracellular tau levels in ATRA-treated cells, which was reversed by lysosome or proteasome inhibitors, suggesting the involvement of both degradation pathways. Our findings highlight the potential therapeutic role of ATRA in Alzheimer's disease and related tauopathies. By enhancing Hsp90 expression and facilitating tau degradation, ATRA could contribute to the clearance of pathological tau proteins, offering a promising strategy for mitigating neurodegeneration. This research underscores the need for further exploration into the molecular mechanisms of tau protein internalization and degradation, which could provide valuable insights into the treatment of neurodegenerative diseases.

Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism

Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.

Abstract IA015: Identifying and targeting synthetic lethalities of aneuploid (cancer) cells

Abstract Aneuploidy, an imbalanced number of chromosomes or chromosome arms, is a genetic hallmark of cancer cells, yet aneuploidy remains a biological enigma and a missed opportunity for cancer therapy. Aneuploid cells must cope with several types of cellular stresses, potentially creating synthetic lethalities that can be used to target aneuploid cancer cells. Here, I will describe our efforts to identify synthetic lethalities of the aneuploid state (in contrast to synthetic lethalities of specific recurrent aneuploidies. Specifically, I will focus on three recent unpublished studies, where we report that: (1) Aneuploid cells are preferentially sensitive to perturbation of the spindle assembly checkpoint (SAC) and its regulator KIF18A; the expression and activity of CDC20 determine the sensitivity to SAC inhibition. (2) Aneuploid cells depend on MAPK signaling for overcoming aneuploidy-induced DNA damage; targeting MAPK signaling can sensitize aneuploid cells to DNA damage inducing agents and to PARP inhibitors. (3) Aneuploid cells depend on RNA and protein degradation mechanisms to attenuate the cellular consequence of extra chromosomes; this renders aneuploid cells more sensitive to inhibition of nonsense-mediate decay, miRNA-mediated gene silencing, and the proteasome complex. Citation Format: Uri Ben-David. Identifying and targeting synthetic lethalities of aneuploid (cancer) cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA015.

Proteomics reveals the mechanism of protein degradation and its relationship to sensorial and texture characteristics in dry-cured squid during processing

Proteolysis in dry-cured squid contributes to the development of sensory and textural attributes. In this study, label-free quantitative proteomics was conducted to study the mechanism of proteolysis and its correlation with quality changes. The results showed that the protein profile of dry-cured squid changed markedly during processing, which was confirmed by the quantification of myofibrillar protein, amino nitrogen and total free acids, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Thirty-two key differentially abundant proteins were found to be correlated with sensory and texture characteristics, including myofibrillar protein, tubulin beta chain, collagens, heat shock proteins and cytochrome c. The correlation analysis indicated that myosin regulatory light chain and tubulin beta chain played the most important role in the development of texture and sensory attributes in squid samples during the dry-curing process. The results offered novel insights into proteolysis in dry-cured squid and its relationship to quality changes.

Variant-specific effects of GBA1 mutations on dopaminergic neuron proteostasis.

Glucocerebrosidase 1 (GBA1) mutations are the most important genetic risk factors for Parkinson's disease (PD). Clinically, mild (e.g., p.N370S) and severe (e.g., p.L444P and p.D409H) GBA1 mutations have different PD phenotypes, with differences in age at disease onset, progression, and the severity of motor and non-motor symptoms. We hypothesize that GBA1 mutations cause the accumulation of α-synuclein by affecting the cross-talk between cellular protein degradation mechanisms, leading to neurodegeneration. Accordingly, we tested whether mild and severe GBA1 mutations differentially affect the degradation of α-synuclein via the ubiquitin-proteasome system (UPS), chaperone-mediated autophagy (CMA), and macroautophagy and differentially cause accumulation and/or release of α-synuclein. Our results demonstrate that endoplasmic reticulum (ER) stress and total ubiquitination rates were significantly increased in cells with severe GBA1 mutations. CMA was found to be defective in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons with mild GBA1 mutations, but not in those with severe GBA1 mutations. When examining macroautophagy, we observed reduced formation of autophagosomes in cells with the N370S and D409H GBA1 mutations and impairments in autophagosome-lysosome fusion in cells with the L444P GBA1 mutation. Accordingly, severe GBA1 mutations were found to trigger the accumulation and release of oligomeric α-synuclein in iPSC-derived dopaminergic neurons, primarily as a result of increased ER stress and defective macroautophagy, while mild GBA1 mutations affected CMA, which is mainly responsible for the degradation of the monomeric form of α-synuclein. Overall, our findings provide new insight into the molecular basis of the clinical variability in PD associated with different GBA1 mutations.

Pyrolysis of plasticized films based on soy protein, denatured by different substances

Polymer films based on soy protein isolate (SPI) were obtained using the thermo-pressing method. Plasticizers and denaturing agents were added to ensure better the film-forming properties and reduce the fragility of the resulting materials. Glycerol, sorbitol and their mixture were used for plasticization, while solutions of alkali (potassium hydroxide), surfactants of natural origin (sodium coco sulfate) and reducing agent (sodium sulfite) were used for denaturation. By combining different types of plasticization and denaturation, a series of samples were obtained and compared with a sample based on raw soy protein. In addition to the obvious differences in the appearance of the films, the processes of their thermal degradation, studied by pyrolysis mass spectrometry, also differed significantly. In fact, unprocessed soy protein has the highest thermal stability with the temperature of the most intensive decomposition equal to 270 °С, which can decrease to 200 °С under the conditions of denaturation and plasticization. Despite the increase in the number of film components, the amount of volatile decomposition products decreases (from 86 to 32), as well as the molecular weight of the heaviest of them (from 169 to 74). This is a sign of a change in the mechanism of soy protein degradation due to denaturation and plasticization caused by transformations in its supramolecular structure, such as unfolding and extension of macrochains with increased availability of functional groups. The pyrolytic behavior of some protein samples plasticized with sorbitol is closest to that of untreated soy protein, which may indicate a lower plasticizing efficiency of sorbitol with longer molecules than glycerol. The interpretation of the recorded mass spectra of the volatile pyrolysis products showed that the thermal degradation of protein materials is dominated by processes such as decarboxylation, dehydration, deamination and decarbonylation, while in the presence of plasticizers the splitting of their own molecules also becomes dominant. The characteristic mass spectra of protein films denatured by surfactants also contain ionic fragments of relatively high molecular weight, probably derived from sodium coco sulfate molecules.

The Potential Strategies for Overcoming Multidrug Resistance and Reducing Side Effects of Monomer Tubulin Inhibitors for Cancer Therapy.

Tubulin is an essential target in tumor therapy, and this is attributed to its ability to target MT dynamics and interfere with critical cellular functions, including mitosis, cell signaling, and intracellular trafficking. Several tubulin inhibitors have been approved for clinical application. However, the shortcomings, such as drug resistance and toxic side effects, limit its clinical application. Compared with single-target drugs, multi-target drugs can effectively improve efficacy to reduce side effects and overcome the development of drug resistance. Tubulin protein degraders do not require high concentrations and can be recycled. After degradation, the protein needs to be resynthesized to regain function, which significantly delays the development of drug resistance. Using SciFinder® as a tool, the publications about tubulin-based dual-target inhibitors and tubulin degraders were surveyed with an exclusion of those published as patents. This study presents the research progress of tubulin-based dual-target inhibitors and tubulin degraders as antitumor agents to provide a reference for developing and applying more efficient drugs for cancer therapy. The multi-target inhibitors and protein degraders have shown a development prospect to overcome multidrug resistance and reduce side effects in the treatment of tumors. Currently, the design of dual-target inhibitors for tubulin needs to be further optimized, and it is worth further clarifying the detailed mechanism of protein degradation.

Abstract 6052: Systematic identification of novel targeted protein degradation mechanisms using SITESEEKER® technology

Abstract The rise of Targeted Protein Degradation (TPD) is rapidly changing perceptions about therapeutic target druggability and is rewriting many preconceived notions on drug design. However, the majority of monovalent molecular glue and heterobifunctional degraders in clinical and preclinical development rely predominantly on the recruitment of a single E3 ligase, Cereblon. Arising resistance, toxicities and restricted Protein-of-Interest scope may limit clinical potential, highlighting the need to uncover novel TPD mechanisms. To expand the potential of this modality, we have developed SITESEEKER®, a screening technology operating at substantially greater magnitude of complexity than extant target discovery platforms. SITESEEKER® utilizes computationally-derived encoded mini-protein fragments with huge shape diversity to systematically identify novel degrader mechanisms and define functionally-active binding sites on targets. SITSEEKER® allows for the discovery of targets that may be missed through traditional gene editing or knockdown approaches and can additionally provide valuable mechanistic insights that help to inform, unlock and truncate the path from target ID to drug discovery. We describe the identification of a cache of degrader motifs which showcase the breadth of proteome space yet to be explored within TPD. Moreover, we identify peptide motifs capable of driving degradation in a selective manner with potential to be translated into tissue- or cancer-selective degraders. The functional dependencies of prioritized degraders have been mapped to their cognate E3 ligase using combinatorial screening, giving rise to a number of E3 ligases with potential to be hijacked for TPD. PhoreMost is progressing a pipeline of monovalent and heterobifunctional oncology degrader programs arising from its platform. We demonstrate the effective hijacking of selected E3 ligases, identified by SITESEEKER®, through the discovery of high affinity small molecule binders and subsequent discovery of efficient heterobifunctional degraders against selected Proteins-of-Interest. Citation Format: Christian Dillon. Systematic identification of novel targeted protein degradation mechanisms using SITESEEKER® technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6052.

Insights into the fish protein degradation induced by the fish-borne spoiler Pseudomonas psychrophila and Shewanella putrefaciens: From whole genome sequencing to quality changes

The aim of this study is evaluating the protein degradation capacity of specific spoilage organisms (SSOs) Pseudomonas psychrophila and Shewanella putrefaciens in fish flesh during chilled storage and revealing the underlying genes by whole-genome sequencing (WGS). Biochemical and physical tests were performed on fish flesh inoculated with P. psychrophila and S. putrefaciens individually, including textural properties, myofibrillar fragmentation index, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles, free amino acid composition, total volatile basic nitrogen (TVB-N), trichloroacetic acid (TCA) soluble peptides, and muscle microstructure. Results showed that P. psychrophila and S. putrefaciens exhibited a strong capacity for decomposing the fish protein, and the deterioration of fish flesh texture was primarily attributed to P. psychrophila. The genes from SSOs associated with the production of proteases were identified by whole genome sequencing and serine protease may be the primary enzyme secreted by SSOs involved in the degradation of fish protein. Therefore, the present study has shed light on the mechanisms of protein degradation induced by SSOs, thereby offering valuable insights for the development of effective quality control strategies.

Abstract B033: Functional characterization of ALK5 (TGFBR1) mutations in endometrial cancer

Abstract ALK5 (TGFBR1) is a transmembrane receptor serine/threonine kinase that transduces TGF-b (Transforming Growth Factor b) signaling to activate SMAD2/3-dependent and -independent pathways. The purpose of this study is to determine the functional effects of ALK5 mutations in endometrial cancer (EC). Using nine in silico algorithms, we found that 79% (23 of 39) of ALK5 kinase domain mutations in EC are predicted to impact protein function. To test these predictions experimentally, constructs expressing wildtype-, constitutively active-, kinase-dead, or mutant-ALK5, including ALK5-A230V, were transfected into NIH3T3 cells, which have low endogenous ALK5 levels. Following TGF-β stimulation, we observed that transient exogenous expression of ALK5-A230V, located in the ATP-binding pocket, delayed SMAD2/3 signal transduction and altered SMAD-independent signaling. We further showed that the ALK5-A230V mutant has reduced protein stability via a ubiquitin-dependent protein degradation mechanism. Our structural modeling predicts that SB431542, a small molecule ATP-competitive inhibitor of ALK5 will bind to the ALK5-A230V mutant with less affinity than to wildtype ALK5. We, therefore, examined the inhibitory effect of SB431542 on wildtype- and mutant-ALK5 activity using a Smad-binding element (SBE) luciferase reporter assay in combination with TGF-β stimulation. SBE luciferase activity in ALK5-A230V-transfected cells was inhibited less by SB431542 than in wildtype-ALK5 transfected cells indicating that ALK5-A230V is less sensitive to SB431542 than wildtype ALK5, potentially due to the changes in affinity. Our findings are novel and show that the ALK5-A230V mutant is a partial loss-of-function mutant that attenuates TGF-b signal transduction and has reduced sensitivity to an ALK5 small molecule inhibitor. Citation Format: Eun-Jeong Yu, Daphne W. Bell. Functional characterization of ALK5 (TGFBR1) mutations in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B033.

Metagenomic insights into protein degradation mechanisms in natural fermentation of cassava leaves

Cassava (Manihot esculenta Crantz) leaves, the primary by-product of cassava processing, constitute a significant protein source, accounting for 18 to 38 percent on a dry weight basis. Despite their nutritional value, a substantial portion of these leaves is often discarded post-harvest, resulting in notable resource waste. This study employs metagenomic technology to investigate the protein degradation mechanism in cassava leaves, aiming to provide a technical reference for value-added of this by-product. Following a 36-hour period of natural fermentation, the protein degradation rate reached 58%, a phenomenon intricately linked to both the microbial community structure and its functional properties. Notably, Lactococcus and Enterobacter, recognized for their abundant protease activity, were predominant. Metagenomically assembled genomes further revealed Lactococcus’s substantial role in producing flavors and active compounds, including amino acids and peptides. This study offers novel perspectives to the foodization and high-value utilization of cassava by-products, emphasizing the sustainable exploitation of biomass resources.

Integrated proteome and physiological traits reveal interactive mechanisms of new leaf growth and storage protein degradation with mature leaves of evergreen citrus trees.

The growth of fruit trees depends on the nitrogen (N) remobilization in mature tissues and N acquisition from the soil. However, in evergreen mature citrus (Citrus reticulata Blanco) leaves, proteins with N storage functions and hub molecules involved in driving N remobilization remain largely unknown. Here, we combined proteome and physiological analyses to characterize the spatiotemporal mechanisms of growth of new leaves and storage protein degradation in mature leaves of citrus trees exposed to low-N and high-N fertilization in the field. Results show that the growth of new leaves is driven by remobilization of stored reserves, rather than N uptake by the roots. In this context, proline and arginine in mature leaves acted as N sources supporting the growth of new leaves in spring. Time-series analyses with gel electrophoresis and proteome analysis indicated that the mature autumn shoot leaves are probably the sites of storage protein synthesis, while the aspartic endopeptidase protein is related to the degradation of storage proteins in mature citrus leaves. Furthermore, bioinformatic analysis based on protein-protein interactions indicated that glutamate synthetase and ATP-citrate synthetase are hub proteins in N remobilization from mature citrus leaves. These results provide strong physiological data for seasonal optimization of N fertilizer application in citrus orchards.

CAND1 inhibits Cullin-2-RING ubiquitin ligases for enhanced substrate specificity.

Through targeting essential cellular regulators for ubiquitination and serving as a major platform for discovering proteolysis-targeting chimera (PROTAC) drugs, Cullin-2 (CUL2)-RING ubiquitin ligases (CRL2s) comprise an important family of CRLs. The founding members of CRLs, the CUL1-based CRL1s, are known to be activated by CAND1, which exchanges the variable substrate receptors associated with the common CUL1 core and promotes the dynamic assembly of CRL1s. Here we find that CAND1 inhibits CRL2-mediated protein degradation in human cells. This effect arises due to altered binding kinetics, involving CAND1 and CRL2VHL, as we illustrate that CAND1 dramatically increases the dissociation rate of CRL2s but barely accelerates the assembly of stable CRL2s. Using PROTACs that differently recruit neo-substrates to CRL2VHL, we demonstrate that the inhibitory effect of CAND1 helps distinguish target proteins with different affinities for CRL2s, presenting a mechanism for selective protein degradation with proper pacing in the changing cellular environment.

Ubiquitin Signaling in the Immune System.

Protein ubiquitination is a post-translational modification of proteins that is widespread in eukaryotic cells. It was identified initially while investigating the mechanisms of intracellular protein degradation. There is mounting evidence that ubiquitination and its reverse process, deubiquitination, play a critical regulatory role in the process of intrinsic and adaptive immune responses by regulating the function of various immune cell types, thereby influencing the development of a variety of major human diseases such as autoimmune diseases, infectious diseases, and malignancies. This article will discuss current advances in protein ubiquitination-mediated control of several immune cell functions and its significance in multiple sclerosis (MS).

Quality changes of whitespotted conger (Conger myriaster) based physicochemical changes and label-free proteomics analysis during frozen storage

Whitespotted conger (Conger myriaster) muscle proteins were susceptible to oxidative denaturation during frozen storage. The objective of this study was to investigate the alterations in quality through physicochemical analysis and proteomics after whitespotted conger stored at temperatures of −18 °C and −60 °C. The microstructural observation revealed the noticeable variations such as increased interstitial space and fractured muscle fibre with extension of frozen storage time, and the muscle fibre of whitespotted conger stored at −60 °C were more intact than those stored at −18 °C. The raised TVB-N value indicated that the freshness of whitespotted conger decreased during 120-day frozen storage period. Analysis of myofibrillar protein content and SDS-PAGE demonstrated that compared to −18 °C, lower storage temperature (−60 °C) could better maintain the structure of whitespotted conger muscle by inhibiting protein degradation and oxidation. To reveal the mechanism of protein degradation, label-free quantitative proteomic analysis was performed through LC-MS/MS. The structural proteins including domain-associated proteins and actin-related proteins were up-regulated during frozen storage, but the phosphoglycerate kinase, phosphoglycerate mutase, and fructose-bisphosphate aldolase were down-regulated. Storage at −18 °C accelerated the up- or down-regulation of those differentially abundant proteins. According to KEGG analysis, up- or down-regulated pathways such as glycolysis/gluconeogenesis, carbon metabolism, biosynthesis of amino acids, and calcium signalling pathway mainly accounted for the protein degradation and quality reduction of whitespotted conger at low temperature. These results provided a theoretical basis for improving the quality stability of whitespotted conger during frozen storage.