Urinary Protein Post-Translational Modifications in Patients with Metabolic-Associated Fatty Liver Disease (MAFLD)

Introduction

Metabolic-associated fatty liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has become the most prevalent chronic liver disease worldwide. Its pathogenesis is complex, involving insulin resistance, oxidative stress, and inflammatory responses. Recent studies have highlighted that post-translational modifications (PTMs) of proteins play a critical role in the progression of MAFLD by regulating metabolic pathways and cellular signaling. While much research has focused on hepatic tissue, urinary protein PTMs offer a non-invasive window into the systemic metabolic state of these patients.

The Significance of Protein Post-Translational Modifications

Post-translational modifications, such as phosphorylation, acetylation, ubiquitination, and glycosylation, significantly expand the functional diversity of the proteome. In the context of MAFLD, aberrant PTM patterns can alter the activity of key enzymes involved in lipid metabolism. For instance, the acetylation status of mitochondrial proteins often dictates the efficiency of fatty acid $\beta$-oxidation. Understanding these modifications is essential for identifying novel biomarkers and therapeutic targets.

Urinary Proteomics and PTM Analysis in MAFLD

Urinary proteomics has emerged as a powerful tool for clinical diagnostics due to the ease of sample collection and the relative stability of urinary proteins. In patients with MAFLD, the urinary PTM profile reflects not only renal function but also systemic metabolic dysregulation and liver-derived signaling molecules.

[TABLE:1]

Advanced mass spectrometry techniques have enabled the high-throughput identification of modified peptides in urine. Research indicates that specific phosphorylation patterns in urinary proteins are correlated with the severity of hepatic steatosis and fibrosis. Furthermore, changes in the glycan structures of urinary glycoproteins have been observed in patients transitioning from simple steatosis to metabolic-associated steatohepatitis (MASH).

Mechanisms Linking Urinary PTMs to MAFLD Progression

The presence of specific PTMs in urine is often a consequence of the "multiple-hit" hypothesis of MAFLD. Chronic inflammation leads to the activation of various kinases, which in turn modify downstream targets that may eventually be excreted or filtered into the urine. For example, the modification $\mathcal{M}_{ptm}$ can be represented as a function of the metabolic stress $\sigma$ and the inflammatory index $\phi$:

$$\mathcal{M}_{ptm} = f(\sigma, \phi, \Delta t)$$

where $\Delta t$ represents the progression time of the metabolic disorder.

Abstract

Metabolic-associated fatty liver disease (MAFLD) is a highly prevalent chronic liver disease worldwide, and its progression poses significant health risks. This study utilizes publicly available raw urinary proteomic data to perform a comparative analysis of the differential characteristics of urinary protein post-translational modifications (PTMs) among healthy controls, patients with mild hepatic steatosis (as measured by MRI-PDFF), and patients with severe hepatic steatosis.

The results revealed 144 differential modifications between the mild steatosis group and the healthy control group, 235 differential modifications between the severe steatosis group and the healthy control group, and 181 differential modifications between the mild and severe steatosis groups. Among these, several proteins with differential modifications have been previously reported to play functional roles or undergo changes during the progression of MAFLD. Furthermore, 12 specific proteins exhibited simultaneous changes in both expression levels and modification states across the mild and severe groups. These findings demonstrate that the urinary proteome of patients with mild and severe hepatic steatosis differs significantly from that of healthy individuals, providing a new perspective for non-invasive diagnosis and mechanistic exploration.

Keywords: Metabolic-associated Fatty Liver Disease (MAFLD), Urinary Proteomics, Post-translational Modifications (PTMs)

1 Introduction

Metabolic-associated fatty liver disease (MAFLD) is the most prevalent chronic liver disease globally. Its core definition involves intrahepatic lipid accumulation exceeding 5% of the liver weight—after excluding factors such as alcohol abuse and viral hepatitis—and is directly linked to metabolic abnormalities such as insulin resistance or type 2 diabetes. Since its initial discovery in 1980, its global prevalence has risen sharply, currently affecting approximately 25% of the adult population. The burden of MAFLD among adolescents and young adults has increased significantly, with global estimates of prevalence rising by 75.31%, making it a major public health challenge. The progression of MAFLD is notably hazardous; it can advance from simple hepatic steatosis to metabolic-associated steatohepatitis (MASH), further progress to liver fibrosis, and even increase the risk of hepatocellular carcinoma (HCC).

Beyond the liver, MAFLD affects multiple organ systems, correlating with an increased incidence of cardiovascular disease and chronic kidney disease. The primary lipid abnormality in MAFLD is elevated triglycerides, and its pathological mechanisms involve multi-faceted dysfunctions, including imbalances in hepatic lipid metabolism and increased production of reactive oxygen species (ROS). Unlike blood, urine is not regulated by systemic homeostatic mechanisms, allowing it to more sensitively capture dynamic changes in physiological and pathological states. Urinary proteomics, with its advantages of non-invasive sampling and the capacity for continuous monitoring, provides critical technical support for the systematic analysis of these processes.

Protein post-translational modification (PTM) refers to the enzymatic addition of chemical groups to proteins after synthesis. PTMs regulate key biological processes, such as signal transduction and metabolic pathways, by altering protein structure and activity. Abnormal PTMs have been confirmed to be closely related to the progression of MAFLD. For instance, phosphorylation of AMPK at the Thr172 site activates lipid oxidation, while phosphorylated NF-kB drives inflammatory cytokine production. However, systematic studies of the urinary proteome and PTMs in MAFLD patients remain scarce.

Currently, the severity assessment of MAFLD primarily relies on imaging methods. Magnetic resonance imaging proton density fat fraction (MRI-PDFF) can accurately assess hepatic triglyceride content. Clinically, an MRI-PDFF value between 5% and 15% is typically defined as mild hepatic steatosis, while a value greater than 20% is defined as severe steatosis. By comparing the urinary proteomes and protein modification changes of healthy individuals with MAFLD patients classified by MRI-PDFF, this study aims to explore protein modification changes across different stages of the disease.

2 Materials and Methods

2.1 Data Sources and Sample Information

The raw urinary proteomic data for the MAFLD and healthy control groups were obtained from a cross-sectional study conducted at West China Hospital, Sichuan University (ProteomeXchange accession: PXD026333). Participants were categorized into three groups based on MRI-PDFF results: a healthy control group, a mild hepatic steatosis group (MRI-PDFF $\geq 5\%$), and a severe hepatic steatosis group (MRI-PDFF $\geq 14\%$). Participants with other liver diseases, renal insufficiency, or cancer were excluded.

2.2 Sample Preparation and Mass Spectrometry Acquisition

Mid-stream morning urine was centrifuged and concentrated using ultrafiltration. Samples underwent alkylation and double enzymatic digestion using trypsin and Lys-C. Digested peptides were separated using an EASY-nLC system, and data-independent acquisition (DIA) was performed on a FAIMS-equipped Orbitrap Exploris mass spectrometer.

2.3 Unrestricted Modification Search (Open-pFind)

An unrestricted modification search was performed on the raw data using pFind Studio software against the UniProt human protein database. The instrument type was configured as HCD-FTMS. Trypsin was selected as the enzyme with full cleavage specificity, allowing for two missed cleavages. The search was conducted in open search mode with a peptide-level false discovery rate (FDR) threshold set to 1%.

2.4 Bioinformatics Analysis of PTMs

Following the open modification search, the Python script pFind_protein_contrast_script was utilized to integrate modification data across samples. Intergroup comparisons were conducted to screen for differentially modified proteins (DMPs) using criteria of a fold change (FC) $\ge 1.2$ or $\le 0.83$ and a $p$-value $< 0.05$ (heteroscedastic independent $t$-test). Functional annotation was performed using the UniProt database and PubMed literature retrieval.

3 Results

3.1 Comparative Analysis: Mild Steatosis vs. Healthy Control

Comparing the urinary PTMs of the mild hepatic steatosis group with the healthy control group revealed 281 differentially modified sites. Several proteins harboring these PTMs are associated with lipid metabolism. For instance, Glutathione hydrolase proenzyme (P19440, $p = 3.82\text{E-}02$) and Alpha-1-acid glycoprotein (P19652, $p = 4.65\text{E-}02$) were significantly correlated with the degree of hepatic steatosis. Additionally, Galectin-3-binding protein (Q08380, $p = 4.88\text{E-}02$) and Aminoacylase-1 (Q03154, $p = 3.54\text{E-}02$) showed increased expression or modification in the mild group. Notably, modifications to BHMT2 (Q9H2M3) were detected exclusively in the mild hepatic steatosis group.

3.2 Comparative Analysis: Severe Steatosis vs. Healthy Control

The comparison between the severe hepatic steatosis group and the healthy group revealed 445 differentially modified sites. Key proteins included L-lactate dehydrogenase B chain (P07195), where acetylation is linked to impaired hepatic lactate clearance. Deficiency in Vitronectin (P04004, $p = 4.02\text{E-}03$) has been shown to alleviate liver fibrosis. Other significant proteins included Sialidase-1 (Q99519, $p = 2.51\text{E-}02$), Fibronectin (P02751, $p = 3.88\text{E-}02$), and Lactotransferrin (P02788, $p = 4.37\text{E-}02$). Modifications of proteins such as Ceruloplasmin (P00450) and Albumin (P02768) were identified only in the severe group.

3.3 Comparative Analysis: Mild vs. Severe Steatosis

Significant differences were found between the mild and severe hepatic steatosis groups. Tissue alpha-L-fucosidase (P04066, $p = 3.83\text{E-}02$) levels were positively correlated with metabolic syndrome components. Other candidates included Resistin (Q9HD89, $p = 4.99\text{E-}02$), which increases with liver injury progression, and Osteopontin (P10451, $p = 3.61\text{E-}02$), which regulates inflammatory responses. Modification of Phosphoinositide-3-kinase-interacting protein 1 (Q96FE7) was significantly present in the severe group but absent in the mild group.

3.4 Analysis of Key MAFLD Regulatory Proteins

Analysis of proteins such as AMPK, SREBP1c, and PNPLA3 revealed that while PTMs were detected across all groups, the specific sites differed from previously reported functional modifications (e.g., AMPK Thr172). The open modification search primarily identified basic types like deamidation and pyroglutamination, while functional modifications directly involved in MAFLD regulation were not detected in the urinary proteome.

3.5 Comparison of Differential Expression and Modification

A cross-study correlation analysis compared differentially expressed proteins (DEPs) from existing literature with the differentially modified proteins (DMPs) from this study. Six proteins exhibited significant changes in both expression and modification: Thyroxine-binding globulin, Immunoglobulin heavy constant gamma 1, Peptidase inhibitor 16, Ceruloplasmin, Alpha-1B-glycoprotein, and Alpha-1-acid glycoprotein 1. This suggests these proteins are key molecular nodes in MAFLD progression.

4 Discussion

This study demonstrates that urinary protein modification profiles are correlated with the severity of MAFLD. We identified 144 differential modifications in the mild group and 235 in the severe group compared to controls. The heterogeneity of these modifications across pathological stages suggests their potential as non-invasive biomarkers.

The discrepancy between the modifications identified here and previously reported functional sites (such as those on SREBP1c) may be due to the nature of urinary proteins, which are products of systemic metabolism and may reflect degradation or transport states rather than primary hepatic signaling. Furthermore, the functional annotation of many identified PTMs remains incomplete in the context of MAFLD. Nevertheless, the enrichment of specific modification types reflects the body's systemic response to hepatic metabolic disorders.

5 Conclusion

We systematically analyzed the differential modification characteristics of the urinary proteome in MAFLD patients. The identification of a significant number of MAFLD-related proteins with altered PTMs provides a new perspective for non-invasive diagnosis and the exploration of disease mechanisms. Future research with larger, multi-center cohorts is needed to validate these findings for clinical translation.