The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair.

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.

Implications of inhibition of Rev1 interaction with Y family DNA polymerases for cisplatin chemotherapy.

Chemotherapy with cisplatin becomes limiting due to toxicity and secondary malignancies. In principle, therapeutics could be improved by targeting translesion synthesis (TLS) polymerases (Pols) that promote replication through intrastrand cross-links, the major cisplatin-induced DNA adduct. However, to specifically target malignancies with minimal adverse effects on normal cells, a good understanding of TLS mechanisms in normal versus cancer cells is paramount. We show that in normal cells, TLS through cisplatin intrastrand cross-links is promoted by Polη- or Polι-dependent pathways, both of which require Rev1 as a scaffolding component. In contrast, cancer cells require Rev1-Polζ. Our findings that a recently identified Rev1 inhibitor, JH-RE-06, purported to specifically disrupt Rev1 interaction with Polζ to block TLS through cisplatin adducts in cancer cells, abrogates Rev1's ability to function with Y family Pols as well, implying that by inactivating Rev1-dependent TLS in normal cells, this inhibitor will exacerbate the toxicity and tumorigenicity of chemotherapeutics with cisplatin.

SpotLink enables sensitive and precise identification of site nonspecific cross-links at the proteome scale.

Nonspecific cross-linker can provide distance restraints between surface residues of any type, which could be used to investigate protein structure construction and protein-protein interaction (PPI). However, the vast number of potential combinations of cross-linked residues or sites obtained with such a cross-linker makes the data challenging to analyze, especially for the proteome-wide applications. Here, we developed SpotLink software for identifying site nonspecific cross-links at the proteome scale. Contributed by the dual pointer dynamic pruning algorithm and the quality control of cross-linking sites, SpotLink identified > 3000 cross-links from human cell samples within a short period of days. We demonstrated that SpotLink outperformed other approaches in terms of sensitivity and precision on the datasets of the simulated succinimidyl 4,4'-azipentanoate dataset and the condensin complexes with known structures. In addition, some valuable PPI were discovered in the datasets of the condensin complexes and the HeLa dataset, indicating the unique identification advantages of site nonspecific cross-linking. These findings reinforce the importance of SpotLink as a fundamental characteristic of site nonspecific cross-linking technologies.

Enzymatic and Non-enzymatic Collagen Cross-Links and Fracture Occurrence in Type 1 Diabetes Patients.

Increased fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD) by DXA. Advanced glycation end-products (AGEs) have been implicated in the increased fracture risk in T1D, yet recent publications question this. To test the hypothesis that enzymatic collagen cross-links rather than AGEs correlate with fracture incidence in T1D, we analyzed iliac crest biopsies from sex-matched, fracturing T1D patients (N = 5; T1DFx), 6 non-fracturing T1D patients (T1DNoFx), and 6 healthy subjects, by Raman microspectroscopy as a function of tissue age (based on double fluorescent labels), in intracortical and trabecular bone, to determine pyridinoline (Pyd), ε-N-Carboxymethyl-L-lysine, and pentosidine (PEN)). There were no differences in the clinical characteristics between the T1DFx and T1DNoFx groups. At trabecular forming surfaces, T1DFx patients had higher PEN and Pyd content compared to T1DNoFx ones. Previous studies have shown that elevated PEN does not necessarily correlate with fracture incidence in postmenopausal, long-term T1D patients. On the other hand, the elevated Pyd content in the T1DFx patients would be consistent with published studies showing a significant correlation between elevated trivalent enzymatic collagen cross-links and fracture occurrence independent of BMD. Collagen fibers with high Pyd content are more brittle. Thus, a plausible suggestion is that it is the enzymatic collagen cross-links that either by themselves or in combination with the adverse effects of increased AGE accumulation that result in fragility fracture in T1D.

OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria.

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.

MUS81 cleaves TOP1-derived lesions and other DNA-protein cross-links.

BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

Mutations in AcrR and RNA Polymerase Confer High-Level Resistance to Psoralen-UVA Irradiation.

DNA interstrand cross-links, such as those formed by psoralen-UVA irradiation, are highly toxic lesions in both humans and bacteria, with a single lesion being lethal in Escherichia coli. Despite the lack of effective repair, human cancers and bacteria can develop resistance to cross-linking treatments, although the mechanisms of resistance remain poorly defined. Here, we subjected E. coli to repeated psoralen-UVA exposure to isolate three independently derived strains that were >10,000-fold more resistant to this treatment than the parental strain. Analysis of these strains identified gain-of-function mutations in the transcriptional regulator AcrR and the alpha subunit of RNA polymerase that together could account for the resistance of these strains. Resistance conferred by the AcrR mutation is mediated at least in part through the regulation of the AcrAB-TolC efflux pump. Resistance via mutations in the alpha subunit of RNA polymerase occurs through a still-uncharacterized mechanism that has an additive effect with mutations in AcrR. Both acrR and rpoA mutations reduced cross-link formation in vivo. We discuss potential mechanisms in relation to the ability to repair and survive interstrand DNA cross-links. IMPORTANCE Psoralen DNA interstrand cross-links are highly toxic lesions with antimicrobial and anticancer properties. Despite the lack of effective mechanisms for repair, cells can become resistant to cross-linking agents through mechanisms that remain poorly defined. We derived resistant mutants and identified that two gain-of-function mutations in AcrR and the alpha subunit of RNA polymerase confer high levels of resistance to E. coli treated with psoralen-UVA. Resistance conferred by AcrR mutations occurs through regulation of the AcrAB-TolC efflux pump, has an additive effect with RNA polymerase mutations, acts by reducing the formation of cross-links in vivo, and reveals a novel mechanism by which these environmentally and clinically important agents are processed by the cell.

Characterization of extracellular matrix deposited by segmental trabecular meshwork cells.

PURPOSE: Biophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes. The underlying reason for segmentation or differential response to pressure and stiffening are unknown. This is largely due to a lack of appropriate models (in vitro or ex vivo) to study this phenomena. METHODS: Primary trabecular meshwork cells were isolated from segmental flow regions, and cells were cultured for 4 weeks in the presence or absence or dexamethasone to obtain cell derived matrices (CDM). The biomechanical attributes of the CDM, composition of the matrisome, and incidence of crosslinks were determined by atomic force microscopy and mass spectrometry. RESULTS: Data demonstrate that matrix deposited by cells from low flow regions are stiffer and exhibit a greater number of immature and mature crosslinks, and that these are exacerbated in the presence of steroid. We also show a differential response of high or low flow cells to steroid via changes observed in the matrix composition. However, no correlations were observed between elastic moduli and presence or absence of mature and immature crosslinks in the CDMs. CONCLUSION: Regardless of a direct correlation between matrix stiffness and crosslinks, we observed distinct differences in the composition and mechanics of the matrices deposited by segmental flow cells. These results suggest distinct differences in cellular identify and likely a basis for mechanical memory post isolation and culture. Nevertheless, we conclude that although a mechanistic basis for matrix stiffness was undetermined in this study, it is a viable tool to study cell-matrix interactions and further our understanding of trabecular meshwork pathobiology.

Structural characterisation and pH-dependent preference of pyrrole cross-link isoforms from reactions of oxoenal with cysteine and lysine side chains as model systems.

In this study, we subjected 5,5-diethoxy-4-oxopent-2-enal (DOPE), a model amino acids cross-linking reagent, to reactions with N-acetylcysteine (Ac-Cys) and Nα-acetyllysine (Ac-Lys), and identified three pyrrole cross-links. The compounds were isolated and their structures were rigorously determined by spectrometric and spectroscopic methods, including 2D NMR experiments. The use of 2D NMR spectroscopy was crucial to determine the position of the substituents in the pyrrole rings. The products were identified as 2,4-, 2,3-, and 2,5-substituted pyrroles. The data obtained from their structural characterisation can help similar studies on amino acids modifications induced by analogous bifunctional carbonyl compounds. Our results show that the study of pathways in which model electrophiles modify amino acids may be helpful for similar studies dealing with identification of structural changes in cysteine- and lysine-containing proteins associated with oxidative stress.

Contribution of intramuscular connective tissue and its structural components on meat tenderness-revisited: a review.

The tenderness of meat influences consumers' perceptions of its quality. Meat tenderness is a key quality characteristic that influences consumer satisfaction, repeat purchases, and willingness to pay higher prices for meat. Muscle fibers, connective tissues, and adipocytes are the main structural components of meat that contribute to its tenderness and texture. In the present review, we have focused on the role of connective tissue and its components in meat tenderness, specifically perimysial intramuscular connective tissue (IMCT) and its concept as an immutable "background toughness." The collagen contribution to cooked meat toughness can be altered by animal diet, compensatory growth, slaughter age, aging, and cooking. As well, progressive thickening of the perimysium leads to a progressive increase in shear force values in beef, pork, chicken, and this may occur prior to adipocyte formation as cattle finish in feedlots. Conversely, adipocyte accumulation in the perimysium can decrease cooked meat shear force, suggesting that the contribution of IMCT to meat toughness is complex and driven by both collagen structure and content. This review provides a theoretical foundation of information to modify IMCT components to improve meat tenderness.

Peroxidasin-mediated bromine enrichment of basement membranes.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

On the Nature of Stationary and Time-Resolved Fluorescence Spectroscopy of Collagen Powder from Bovine Achilles Tendon.

This paper presents a more systematic study of steady-state and time-resolved autofluorescence spectroscopy of collagen isolated from bovine Achilles tendon. In steady-state fluorescence measurements, the excitation and emission spectra of collagen powder, recorded at different fluorescence excitation and detection wavelengths, were compared with the fluorescence excitation and emission spectra of the amino acids phenylalanine, tyrosine, and tryptophan, as well as with similar spectra for 13 autofluorescent collagen cross-links, which have been identified and described in the literature so far. In time-resolved studies, fluorescence was excited by the pulsed light of different wavelengths, and for each excitation wavelength, fluorescence decay was recorded for several detection wavelengths. Data analysis allowed recovery of the fluorescence decay times for each experimental excitation detection event. The obtained information on the decay times of the measured fluorescent signals was discussed, taking into account the available literature data from similar studies of isolated collagen and collagen-rich tissues. Based on the obtained results, it was found that the shape and position of the measured fluorescence excitation and emission spectra of collagen strongly depend on the emission and excitation wavelengths selected in the measurements. From the recorded excitation and emission bands of collagen, it can be concluded with high probability that there are additional, so far unidentified, collagen cross-links, which can be excited at longer excitation wavelengths. In addition, the collagen excitation spectra were measured at longer emission wavelengths at which the collagen cross-links emit fluorescent light. In addition to the emission spectra obtained for excitation in the deep-UV region, the results of time-resolved fluorescence studies with excitation in the deep-UV region and detection at longer wavelengths suggest that fluorescence excitation energy transfer processes occur from the amino acids to the collagen cross-links, and also between the cross-links themselves.

Bypass of Abasic Site-Peptide Cross-Links by Human Repair and Translesion DNA Polymerases.

DNA-protein cross-links remain the least-studied type of DNA damage. Recently, their repair was shown to involve proteolysis; however, the fate of the peptide remnant attached to DNA is unclear. Particularly, peptide cross-links could interfere with DNA polymerases. Apurinuic/apyrimidinic (AP) sites, abundant and spontaneously arising DNA lesions, readily form cross-links with proteins. Their degradation products (AP site-peptide cross-links, APPXLs) are non-instructive and should be even more problematic for polymerases. Here, we address the ability of human DNA polymerases involved in DNA repair and translesion synthesis (POLß, POLλ, POLη, POLκ and PrimPOL) to carry out synthesis on templates containing AP sites cross-linked to the N-terminus of a 10-mer peptide (APPXL-I) or to an internal lysine of a 23-mer peptide (APPXL-Y). Generally, APPXLs strongly blocked processive DNA synthesis. The blocking properties of APPXL-I were comparable with those of an AP site, while APPXL-Y constituted a much stronger obstruction. POLη and POLκ demonstrated the highest bypass ability. DNA polymerases mostly used dNTP-stabilized template misalignment to incorporate nucleotides when encountering an APPXL. We conclude that APPXLs are likely highly cytotoxic and mutagenic intermediates of AP site-protein cross-link repair and must be quickly eliminated before replication.

Cross-linking and modification of fibronectin by peroxynitrous acid: Mapping and quantification of damage provides a new model for domain interactions.

Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where it is exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitrotyrosine, di-tyrosine, and 6-nitrotryptophan. We have shown previously that human plasma FN is readily modified by ONOOH, but the extent and location of modifications, and the role of FN structure (compact versus extended) in determining these factors is poorly understood. Here, we provide a detailed LC-MS analysis of ONOOH-induced FN modifications, including the extent of their formation and the sites of intramolecular and intermolecular cross-links, including Tyr-Tyr, Trp-Trp, and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of ECM modification and turnover in the many pathologies associated with inflammation-associated fibrosis.

Phenyl Selenide-Based Precursors as Hydrogen Peroxide Inducible DNA Interstrand Cross-Linkers.

DNA interstrand crosslinks (ICLs) are highly toxic DNA lesions, and induce cell death by blocking DNA strand separation. Most ICL agents aiming to kill cancer cells, also generate adverse side effects to normal cells. H2 O2 -inducible DNA ICL agents are highly selective for targeting cancer cells, as the concentration of H2 O2 is higher in cancer cells than normal cells. Previous studies have focused on arylboronate-based precursors, reacting with H2 O2 to generate reactive quinone methides (QMs) crosslinking DNA. Here we explore phenyl selenide-based precursors 1-3 as H2 O2 -inducible DNA ICL agents. The precursors 1-3 can be activated by H2 O2 to generate the good benzylic leaving group and promote production of reactive QMs to crosslink DNA. Moreover, the DNA cross-linking ability is enhanced by the introduction of substituents in the para-position of the phenolic hydroxyl group. From the substituents explored (H, OMe, F), the introduction of electron donating group (OMe) shows a pronounced elevating effect. Further mechanistic studies at the molecular and DNA levels confirm alkylation sites located mainly at dAs, dCs and dGs in DNA. Additionally, cellular experiments reveal that agents 1-3 exhibit higher cytotoxicity toward H1299 human lung cancer cells compared to clinically used drugs, by inducing cellular DNA damage, apoptosis and G0/G1 cell cycle arrest. This study provides a strategy to develop H2 O2 -inducible DNA interstrand cross-linkers.

Engineering Human Neuroglobin into a Cytochrome c-Like Protein with a Single Thioether Bond in Non-native State.

A double mutant of human H64M/V71C neuroglobin (Ngb) was engineered, which formed a single thioether bond as that in atypical cytochrome c, whereas the heme distal Met64 was oxidized to both sulfoxide (SO-Met) and sulfone (SO2 -Met). By contrast, no Cys-heme cross-link was formed in V71C Ngb with His64/His96 coordination, as shown by the X-ray crystal structure, which indicates that an open distal site facilitates the activation of heme iron for structural modifications.

"Metal-modified base pairs" vs. "metal-mediated pairs of bases": not just a semantic issue!

A "nucleobase pair" is not identical with a "pair of basic ligands", as only in the first case, the existence of inter-base hydrogen bonds is implied. The cross-linking of two nucleobases or two basic ligands by a metal ion of suitable geometry produces either "metal-modified" or "metal-mediated" species, but in the author's opinion, this difference is not always properly made. This commentary is an attempt to provide a clearer distinction between the two scenarios.

Maximum number of bone cross-linked vertebrae: an index for BMD in diffuse idiopathic skeletal hyperostosis.

INTRODUCTION: The maximum number of vertebral bodies with bony bridges between adjacent vertebrae (max VB) helps assess the risk of fracture in diffuse idiopathic skeletal hyperostosis (DISH). In addition to max VB, the maximum thickness of bone cross-bridges (max TB) may be an index of bone mineral density (BMD). Therefore, this study investigated the relationship among max VB, max TB, and BMD. MATERIALS AND METHODS: The participants in this cross-sectional study were male patients (n = 123) with various max VB from the thoracic vertebrae to the sacrum without sacroiliac ankylosis. The participants were grouped by max VB. For example, a group with max VB from 4 to 8 would be listed as max VB (4-8). The relation between femur proximal BMD and mean max TB and max VB was assessed. Femur proximal BMD was then compared after adjusting for confounding factors. RESULTS: The results indicated that max VB was correlated with femur proximal BMD in max VB (0-8) and max VB (9-18) groups. The mean max TB was correlated only with femur proximal BMD in max VB (0-8). After adjusting, max VB (4-8) showed a significantly higher femur proximal BMD than max VB (0-3) and max VB (9-18). CONCLUSION: Femur proximal BMD and mean max TB showed different trends after max VB = 9, which suggests that max VB is an index of BMD, and that DISH has at least two possible populations in terms of BMD and bone cross-link thickness.

New insight into the molecular mechanism of protein cross-linking induced by cis-2-butene-1,4-dial, the metabolite of furan: Formation of 2-substituted pyrrole cross-links involving the cysteine and lysine residues.

Furan is an environmental pollutant also present in heat-treated food. The compound is toxic and carcinogenic to rats, and classified as a possible human carcinogen. Mechanisms laying behind the furan carcinogenicity remain unclear, however scientific data indicate the involvement of bioactivation catalysed by cytochrome P450 2E1. The resulted initial metabolite of furan, cis-2-butene-1,4-dial (BDA) is an extremely reactive conjugated dialdehyde able to damage important cellular components such as glutathione, proteins and nucleic acids. Earlier works showed that BDA induces protein cross-linking leading to the formation of modifications containing substituted pyrrole ring, and that the cysteine and lysine residues are prone to undergo such processes. The resulted cross-linked protein adducts are important for research aimed at exploring biomarkers of furan exposure. Due to furan's high volatility, biomarker-based methods appear to be a reliable approach to measure a level of exposure to this chemical. To date, numerous urinary and hepatocyte metabolites were identified. However, all the metabolites contain 3-substituted pyrrole ring resulted from the 1,4-addition of the cysteine thiol function to BDA followed by the condensation with the lysine free amino group. In this work we provide evidence that also 2-substituted pyrrole cross-links are formed at the physiological pH, when BDA is subjected to the reaction with N-acetylcysteine and Nα-acetyllysine, Nε-acetyllysine or lysine, respectively. The 2-substituted cross-links arise from the initial 1,2-addition of N-acetylcysteine to BDA accomplished by the condensation with lysine or its N-acetylated derivatives. Formation of the 2-substituted pyrroles sheds new light on the reactivity of cis-2-butene-1,4-dial towards amino acids, and contributes to complete characterisation of the compound chemistry. Our studies show that the 2- and 3-substituted cross-links are indistinguishable based on their MS and MS/MS spectra, and that for reliable qualitative and quantitative measurements the use of isotope-substituted synthetic internal standards is necessary. In the light of these facts, our results are important for research into furan exposure biomarkers.