Ufmylation: Its Role in Protein Folding and ER Stress

Cells rely on intricate systems to maintain protein homeostasis, ensuring that proteins are correctly folded and functional. When this balance is disrupted, misfolded proteins accumulate, leading to cellular stress and disease. One crucial mechanism in proteostasis is UFMylation, a post-translational modification essential for endoplasmic reticulum (ER) function.

Recent research highlights UFMylation’s role in regulating protein folding and mitigating ER stress. Understanding this process provides insight into broader physiological functions and its implications for human health.

Molecular Basis Of UFMylation

UFMylation involves the covalent attachment of ubiquitin-fold modifier 1 (UFM1) to target proteins, influencing their stability, localization, and function. Unlike ubiquitination, which primarily marks proteins for degradation, UFMylation is closely tied to ER homeostasis, particularly in protein folding and quality control. This modification is catalyzed by a dedicated enzymatic cascade that ensures the precise conjugation of UFM1 to specific substrates.

The process begins with UFM1 activation by the E1-like enzyme UBA5, forming a high-energy thioester bond in an ATP-dependent manner. This activated UFM1 is then transferred to the E2 conjugating enzyme UFC1, which interacts with the E3 ligase UFL1 to facilitate substrate specificity. UFL1, in complex with its cofactor DDRGK1, ensures UFM1 is conjugated to proteins involved in ER-associated processes. One well-characterized target is ribosomal protein RPL26, which enhances ribosome-associated protein folding, reducing the burden on ER chaperones and mitigating stress.

Beyond ribosomal regulation, UFMylation affects ER-associated proteins involved in membrane integrity and trafficking newly synthesized polypeptides. Loss of UFMylation leads to ER expansion and misfolded protein accumulation, underscoring its role in ER function. UFM1-specific proteases, such as UFSP2, remove UFM1 from substrates, allowing for reversible regulation. This balance between conjugation and deconjugation ensures UFMylation remains responsive to cellular conditions.

Key Components In The Conjugation Pathway

The UFMylation pathway relies on three primary enzymes: the E1 activating enzyme UBA5, the E2 conjugating enzyme UFC1, and the E3 ligase UFL1. Each plays a distinct role in UFM1 conjugation, ultimately influencing protein function and cellular homeostasis.

UBA5 initiates the process by activating UFM1 in an ATP-dependent manner, forming a high-energy thioester bond. Structural studies show UBA5 contains a unique adenylation domain that ensures specificity in UFM1 activation. Once activated, UFM1 is transferred to UFC1, which carries it for interaction with the E3 ligase. The structural compatibility between UFM1 and UFC1 prevents unintended modifications of non-target proteins.

UFL1, in complex with DDRGK1, plays a central role in substrate selection and UFM1 conjugation. Unlike many ubiquitin E3 ligases, UFL1 operates through a distinct mechanism still under investigation. This complex, primarily localized to the ER membrane, facilitates UFMylation of ribosomal and ER-associated proteins, including RPL26. The interaction between UFL1 and DDRGK1 is particularly important, as mutations in DDRGK1 have been linked to impaired UFMylation and disruptions in protein homeostasis.

Beyond the conjugation machinery, UFM1-specific proteases such as UFSP2 regulate the reversibility of this modification. UFSP2 cleaves UFM1 from substrates, maintaining balance between UFMylation and de-UFMylation. Mutations in UFSP2 have been associated with neurodevelopmental disorders, underscoring the necessity of precise regulation.

Regulation Of Protein Folding And Degradation

Maintaining protein homeostasis in the ER requires both proper folding and elimination of misfolded proteins. UFMylation contributes to this balance by modulating ribosome-associated factors and ER-resident chaperones, optimizing protein synthesis and maturation. By modifying ribosomal proteins involved in co-translational folding, UFMylation enhances nascent polypeptide processing, reducing the burden on ER chaperones such as BiP and GRP94.

Beyond assisting folding machinery, UFMylation influences the degradation of proteins that fail to achieve their functional conformation. Misfolded proteins are targeted for degradation through ER-associated degradation (ERAD), which relies on ubiquitin-proteasome system components. UFMylation optimizes ERAD efficiency by facilitating the recruitment of degradation factors to affected substrates, ensuring terminally misfolded proteins are recognized and retrotranslocated for proteasomal degradation.

In cases where misfolded proteins persist, UFMylation coordinates with autophagy-related mechanisms to remove aggregates that cannot be processed by the proteasome. Studies suggest UFMylation enhances aggregate clearance by promoting interactions between ER membranes and autophagic machinery, helping cells adapt to heightened folding demands. This function is particularly relevant in neurodegenerative disorders characterized by aggregate-prone proteins.

Involvement In ERphagy

When conventional quality control mechanisms fail, ERphagy—a selective form of autophagy—removes damaged ER fragments to maintain cellular function. UFMylation regulates this process by modifying ER-associated proteins, facilitating the recruitment of autophagic machinery to clear excess or damaged ER membranes.

One key target is FAM134B, an ER-phagy receptor that mediates selective degradation of ER components under stress. UFMylation of FAM134B enhances its interaction with LC3, a core autophagy protein, promoting the encapsulation of ER fragments into autophagosomes. This ensures ERphagy proceeds efficiently, preventing the accumulation of dysfunctional ER structures that could disrupt cellular processes.

Significance In Cellular Stress Responses

Cells constantly face stressors such as nutrient fluctuations, oxidative stress, and disruptions in ER function. UFMylation helps cells adapt by modulating protein folding, degradation, and organelle maintenance. Under ER stress conditions, such as increased protein synthesis or environmental toxins, UFMylation regulates the unfolded protein response (UPR). By optimizing ribosomal proteins and ER-associated chaperones, it prevents misfolded protein accumulation and prolonged stress pathway activation.

Beyond protein quality control, UFMylation links ER function to metabolic regulation. Studies show UFMylation activity increases under ER stress, suggesting a feedback mechanism that enhances cellular recovery. The modification of key ER-resident proteins facilitates adaptive responses, allowing cells to restore homeostasis or initiate degradation pathways when necessary. Disruptions in UFMylation have been associated with increased sensitivity to stress, contributing to chronic inflammation and metabolic imbalances.

Possible Links To Human Health

Emerging evidence links UFMylation dysregulation to various human diseases. Given its role in ER integrity and protein quality control, defects in UFMylation have been implicated in neurodegenerative disorders and metabolic syndromes. Genetic mutations affecting UFMylation components have been associated with congenital disorders characterized by developmental defects.

One area of interest is UFMylation’s role in neurodegeneration. Misfolded protein accumulation is a hallmark of diseases such as Alzheimer’s and Parkinson’s, and impaired UFMylation may contribute to defective protein clearance in affected neurons. Experimental models show that loss of UFMylation leads to increased ER stress and reduced autophagic clearance, conditions commonly observed in neurodegenerative pathology. Additionally, mutations in UFMylation-related genes have been linked to hereditary neurodevelopmental disorders, highlighting its importance in neural health.

In metabolic diseases, UFMylation is implicated in pancreatic beta-cell function and insulin sensitivity. Disruptions in UFM1 conjugation impair ER function in insulin-producing cells, contributing to diabetes progression. Additionally, UFMylation plays a role in liver homeostasis, with dysregulation linked to fatty liver disease and other metabolic disorders. As research continues, UFMylation’s relevance to human health is becoming increasingly evident, offering potential therapeutic targets for diseases associated with ER stress and protein misfolding.