The Influence of Body Fat Dynamics on Pulmonary Immune Responses in Murine Tuberculosis: Unraveling Sex-Specific Insights.

The World Health Organization (WHO) highlights a greater susceptibility of males to tuberculosis (TB), a vulnerability attributed to sex-specific variations in body fat and dietary factors. Our study delves into the unexplored terrain of how alterations in body fat influence Mycobacterium tuberculosis (Mtb) burden, lung pathology, immune responses, and gene expression, with a focus on sex-specific dynamics. Utilizing a low-dose Mtb-HN878 clinical strain infection model, we employ transgenic FAT-ATTAC mice with modulable body fat to explore the impact of fat loss (via fat ablation) and fat gain (via a medium-fat diet, MFD). Firstly, our investigation unveils that Mtb infection triggers severe pulmonary pathology in males, marked by shifts in metabolic signaling involving heightened lipid hydrolysis and proinflammatory signaling driven by IL-6 and localized pro-inflammatory CD8+ cells. This stands in stark contrast to females on a control regular diet (RD). Secondly, our findings indicate that both fat loss and fat gain in males lead to significantly elevated (1.6-fold (p ≤ 0.01) and 1.7-fold (p ≤ 0.001), respectively) Mtb burden in the lungs compared to females during Mtb infection (where fat loss and gain did not alter Mtb load in the lungs). This upsurge is associated with impaired lung lipid metabolism and intensified mitochondrial oxidative phosphorylation-regulated activity in lung CD8+ cells during Mtb infection. Additionally, our research brings to light that females exhibit a more robust systemic IFNγ (p ≤ 0.001) response than males during Mtb infection. This heightened response may either prevent active disease or contribute to latency in females during Mtb infection. In summary, our comprehensive analysis of the interplay between body fat changes and sex bias in Mtb infection reveals that alterations in body fat critically impact pulmonary pathology in males. Specifically, these changes significantly reduce the levels of pulmonary CD8+ T-cells and increase the Mtb burden in the lungs compared to females. The reduction in CD8+ cells in males is linked to an increase in mitochondrial oxidative phosphorylation and a decrease in TNFα, which are essential for CD8+ cell activation.

Susceptibility of Fat Tissue to SARS-CoV-2 Infection in Female hACE2 Mouse Model.

The coronavirus disease (COVID-19) is a highly contagious viral illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 has had a catastrophic effect globally causing millions of deaths worldwide and causing long-lasting health complications in COVID-19 survivors. Recent studies including ours have highlighted that adipose tissue can act as a reservoir where SARS-CoV-2 can persist and cause long-term health problems. Here, we evaluated the effect of SARS-CoV-2 infection on adipose tissue physiology and the pathogenesis of fat loss in a murine COVID-19 model using humanized angiotensin-converting enzyme 2 (hACE2) mice. Since epidemiological studies reported a higher mortality rate of COVID-19 in males than in females, we examined hACE2 mice of both sexes and performed a comparative analysis. Our study revealed for the first time that: (a) viral loads in adipose tissue and the lungs differ between males and females in hACE2 mice; (b) an inverse relationship exists between the viral loads in the lungs and adipose tissue, and it differs between males and females; and (c) CoV-2 infection alters immune signaling and cell death signaling differently in SARS-CoV-2 infected male and female mice. Overall, our data suggest that adipose tissue and loss of fat cells could play important roles in determining susceptibility to CoV-2 infection in a sex-dependent manner.

A novel role of ADGRF1 (GPR110) in promoting cellular quiescence and chemoresistance in human epidermal growth factor receptor 2-positive breast cancer.

While G protein-coupled receptors (GPCRs) are known to be excellent drug targets, the second largest family of adhesion-GPCRs is less explored for their role in health and disease. ADGRF1 (GPR110) is an adhesion-GPCR and has an important function in neurodevelopment and cancer. Despite serving as a poor predictor of survival, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We evaluated the effects of ADGRF1 overexpression on tumorigenesis and signaling pathways using two human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC) cell-line models. We also interrogated publicly available clinical datasets to determine the expression of ADGRF1 in various BC subtypes and its impact on BC-specific survival (BCSS) and overall survival (OS) in patients. ADGRF1 overexpression in HER2+ BC cells increased secondary mammosphere formation, soft agar colony formation, and % of Aldefluor-positive tumorigenic population in vitro and promoted tumor growth in vivo. ADGRF1 co-immunoprecipitated with both Gαs and Gαq proteins and increased cAMP and IP1 when overexpressed. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 overexpression. RNA-sequencing and RPPA analysis revealed inhibition of cell cycle pathways with ADGRF1 overexpression, suggesting cellular quiescence, as also evidenced by cell cycle arrest at the G0/1 phase and resistance to chemotherapy in HER2+ BC. ADGRF1 was significantly overexpressed in the HER2-enriched BC compared to luminal A and B subtypes and predicted worse BCSS and OS in these patients. Therefore, ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.

Constitutively active androgen receptor supports the metastatic phenotype of endocrine-resistant hormone receptor-positive breast cancer.

BACKGROUND: Hormone receptor positive (HR+) breast cancer (BCa) is the most frequently diagnosed subtype. Acquired and intrinsic resistance to conventional endocrine therapy (ET) commonly occurs and prompts incurable metastatic disease. Hence, ET-resistant (ET-R) HR+ BCa presents a therapeutic challenge. Previous studies show elevated androgen receptor (AR) that supports resistance to ET tamoxifen and correlates with HR+ BCa metastasis. Yet surprisingly, studies with AR-blocker enzalutamide (Enz) in ET-R HR+ BCa present conflicting results. We now report that a constitutively active, unique from canonical Enz-targeted, AR accumulates in endocrine resistant HR+ BCa cells. METHODS: AR protein profiles in acquired and intrinsic ET-R HR + -BCa were defined with cell-free modification tests, in-house in-vivo SUMOylation assays, and PLA imaging. Genomic activity of native AR and modified-AR mimetic was tested with reporter assays and limited transcriptome analysis. Spheroid growth and migration studies were used to evaluate inhibitory actions of Enz and combinatorial therapy. RESULTS: Sustained higher molecular weight SUMO-modified AR (SUMO-AR) persists in acquired and intrinsic ET-R BCa cell lines. Concurrently, SUMO isoforms and global SUMO-modified proteome also accumulates in the same cell lines. We identified AR as a novel substrate for the SUMO-E3 ligase HSPB1/Hsp27. Independent of ligand, SUMO-AR is resilient to ubiquitin-mediated proteasomal degradation, enriched in the nucleus, readily chromatin-bound, and transcriptionally active. Constitutive SUMO-AR initiates a gene-expression profile that favors epithelial-mesenchymal transition. Enz combined with a SUMO inhibitor attenuates migration and metastatic phenotype of ET-R HR+ BCa. CONCLUSION: Targeting both unmodified and SUMO-modified AR prevents the metastatic progression of HR+ BCa with ET-R. Video abstract.

Circulating tumor cell investigation in breast cancer patient-derived xenograft models by automated immunofluorescence staining, image acquisition, and single cell retrieval and analysis.

BACKGROUND: Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®-CyteFinder® system. METHODS: CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation. RESULTS: The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method. CONCLUSIONS: Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte-CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression.

Adipocyte-released adipomes in Chagas cardiomyopathy: Impact on cardiac metabolic and immune regulation.

Chronic Trypanosoma cruzi infection leads to Chagas cardiomyopathy (CCM), with varying manifestations such as inflammatory hypertrophic cardiomyopathy, arrhythmias, and dilated cardiomyopathy. The factors responsible for the increasing risk of progression to CCM are not fully understood. Previous studies link adipocyte loss to CCM progression, but the mechanism triggering CCM pathogenesis remains unexplored. Our study uncovers that T. cruzi infection triggers adipocyte apoptosis, leading to the release of extracellular vesicles named "adipomes". We developed an innovative method to isolate intact adipomes from infected mice's adipose tissue and plasma, showing they carry unique lipid cargoes. Large and Small adipomes, particularly plasma-derived infection-associated L-adipomes (P-ILA), regulate immunometabolic signaling and induce cardiomyopathy. P-ILA treatment induces hypertrophic cardiomyopathy in wild-type mice and worsens cardiomyopathy severity in post-acute-infected mice by regulating adipogenic/lipogenic and mitochondrial functions. These findings highlight adipomes' pivotal role in promoting inflammation and impairing myocardial function during cardiac remodeling in CD.

Diets Differently Regulate Tumorigenesis in Young E0771 Syngeneic Breast Cancer Mouse Model.

Breast cancer (BC) is the most diagnosed cancer type, accounting for one in eight cancer diagnoses worldwide. Epidemiological studies have shown that obesity is associated with increased risk of BC in post-menopausal women, whereas adiposity reduces the risk of BC in premenopausal women. The mechanistic link between obesity and BC has been examined by combining murine BC models with high-fat diet (HFD) induced obesity. However, the effect of adiposity (not obesity) induced by a short period of HFD consumption on BC pathogenesis is not well understood. In the current study, we examined the effects of different diet compositions on BC pathogenesis using a young E0771 syngeneic BC mouse model fed on either an HFD or regular diet (RD: a low-fat high-carbohydrate diet) for a short period (4 weeks) before implanting mammary tumors in mice. We analyzed the effect of diet composition on the onset of tumor growth, metastasis, and metabolic and immune status in the tumor microenvironment (TME) using various methods including in vivo bioluminescence imaging and immunoblotting analyses. We showed for the first time that a short-term HFD delays the onset of tumorigenesis by altering the immune and metabolic signaling and energy mechanism in the TME. However, RD may increase the risk of tumorigenesis and metastasis by increasing pro-inflammatory factors in the TME in young mice. Our data suggest that diet composition, adipogenesis, and loss of body fat likely regulate the pathogenesis of BC in a manner that differs between young and post-menopausal subjects.

Diets Differently Regulate Pulmonary Pathogenesis and Immune Signaling in Mice during Acute and Chronic Mycobacterium tuberculosis Infection.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection persists as a leading cause of mortality and morbidity globally, especially in developing and underdeveloped countries. The prevalence of TB-DM (diabetes mellitus) is higher in low- and middle-income countries where TB and DM are most prevalent. Epidemiological data suggest that slight obesity reduces the risk of TB, whereas DM increases the risk of pulmonary TB. Diets can alter the levels of body fat mass and body mass index by regulating systemic adiposity. Earlier, using a transgenic Mtb-infected murine model, we demonstrated that loss of body fat increased the risk of pulmonary bacterial load and pathology. In the present study, we investigated whether increased adiposity alters pulmonary pathology and bacterial load using C57BL/6 mice infected with HN878 Mtb strain and fed a medium-fat diet (MFD). We analyzed the effects of MFD on the lung during acute and chronic infections by comparing the results to those obtained with infected mice fed a regular diet (RD). Histological and biochemical analyses demonstrated that MFD reduces bacterial burden by increasing the activation of immune cells in the lungs during acute infection and reduces necrosis in the lungs during chronic infection by decreasing lipid accumulation. Our data suggest that slight adiposity likely protects the host during active TB infection by regulating immune and metabolic conditions in the lungs.

Sex Differences in Cardiac Pathology of SARS-CoV2 Infected and Trypanosoma cruzi Co-infected Mice.

Coronavirus disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; CoV2) is a deadly contagious infectious disease. For those who survive COVID-19, post-COVID cardiac damage greatly increases the risk of cardiomyopathy and heart failure. Currently, the number of COVID-related cases are increasing in Latin America, where a major COVID comorbidity is Chagas' heart disease, which is caused by the parasite Trypanosoma cruzi. However, the interplay between indeterminate Chagas disease and COVID-19 is unknown. We investigated the effect of CoV2 infection on heart pathology in T. cruzi infected mice (coinfected with CoV2 during the indeterminate stage of T. cruzi infection). We used transgenic human angiotensin-converting enzyme 2 (huACE2/hACE2) mice infected with CoV2, T. cruzi, or coinfected with both in this study. We found that the viral load in the hearts of coinfected mice is lower compared to the hearts of mice infected with CoV2 alone. We demonstrated that CoV2 infection significantly alters cardiac immune and energy signaling via adiponectin (C-ApN) and AMP-activated protein kinase (AMPK) signaling. Our studies also showed that increased ß-adrenergic receptor (b-AR) and peroxisome proliferator-activated receptors (PPARs) play a major role in shifting the energy balance in the hearts of coinfected female mice from glycolysis to mitochondrial ß-oxidation. Our findings suggest that cardiac metabolic signaling may differently regulate the pathogenesis of Chagas cardiomyopathy (CCM) in coinfected mice. We conclude that the C-ApN/AMPK and b-AR/PPAR downstream signaling may play major roles in determining the progression, severity, and phenotype of CCM and heart failure in the context of COVID.

Host Metabolic Changes during Mycobacterium Tuberculosis Infection Cause Insulin Resistance in Adult Mice.

Tuberculosis (TB) is a highly infectious bacterial disease that primarily attacks the lungs. TB is manifested either as latent TB infection (LTBI) or active TB disease, the latter posing a greater threat to life. The risk of developing active TB disease from LTBI is three times higher in individuals with type 2 diabetes mellitus (T2DM). The association between TB and T2DM is becoming more prominent as T2DM is rapidly increasing in settings where TB is endemic. T2DM is a chronic metabolic disorder characterized by elevated blood glucose, insulin resistance, and relative insulin deficiency. Insulin resistance and stress-induced hyperglycemia have been shown to be increased by TB and to return to normal upon treatment. Previously, we demonstrated that adipocytes (or fat tissue) regulate pulmonary pathology, inflammation, and Mycobacterium tuberculosis (Mtb) load in a murine model of TB. Metabolic disturbances of adipose tissue and/or adipocyte dysfunction contribute to the pathogenesis of T2DM. Thus, pathological adipocytes not only regulate pulmonary pathology, but also increase the risk for T2DM during TB infection. However, the cellular and molecular mechanisms driving the interaction between hyperglycemia, T2DM and TB remain poorly understood. Here, we report the impact of Mtb infection on the development of insulin resistance in mice fed on a regular diet (RD) versus high-fat diet (HFD) and, conversely, the effect of hyperglycemia on pulmonary pathogenesis in juvenile and adult mouse models. Overall, our study demonstrated that Mtb persists in adipose tissue and that Mtb infection induces irregular adipocyte lipolysis and loss of fat cells via different pathways in RD- and HFD-fed mice. In RD-fed mice, the levels of TNFα and HSL (hormone sensitive lipase) play an important role whereas in HFD-fed mice, ATGL (adipose triglyceride lipase) plays a major role in regulating adipocyte lipolysis and apoptosis during Mtb infection in adult mice. We also showed that Mtb infected adult mice that were fed an RD developed insulin resistance similar to infected adult mice that were overweight due to a HFD diet. Importantly, we found that a consequence of Mtb infection was increased lipid accumulation in the lungs, which altered cellular energy metabolism by inhibiting major energy signaling pathways such as insulin, AMPK and mToR. Thus, an altered balance between lipid metabolism and glucose metabolism in adipose tissue and other organs including the lungs may be an important component of the link between Mtb infection and subsequent metabolic syndrome.

NPY1R exerts inhibitory action on estradiol-stimulated growth and predicts endocrine sensitivity and better survival in ER-positive breast cancer.

G Protein-Coupled Receptors (GPCRs) represent the largest superfamily of cell-surface proteins. However, the expression and function of majority of GPCRs remain unexplored in breast cancer (BC). We interrogated the expression and phosphorylation status of 398 non-sensory GPCRs using the landmark BC proteogenomics and phosphoproteomic dataset from The Cancer Genome Atlas. Neuropeptide Y Receptor Y1 (NPY1R) gene and protein expression were significantly higher in Luminal A tumors versus other BC subtypes. The trend of NPY1R gene, protein, and phosphosite (NPY1R-S368s) expression was decreasing in the order of Luminal A, Luminal B, Basal, and human epidermal growth factor receptor 2 (HER2) subtypes. NPY1R gene expression increased in response to estrogen and reduced with endocrine therapy in estrogen receptor-positive (ER+) BC cells and xenograft models. Conversely, NPY1R expression decreased in ER+ BC cells resistant to endocrine therapies (estrogen deprivation, tamoxifen, and fulvestrant) in vitro and in vivo. NPY treatment reduced estradiol-stimulated cell growth, which was reversed by NPY1R antagonist (BIBP-3226) in ER+ BC cells. Higher NPY1R gene expression predicted better relapse-free survival and overall survival in ER+ BC. Our study demonstrates that NPY1R mediates the inhibitory action of NPY on estradiol-stimulated growth of ER+ BC cells, and its expression serves as a biomarker to predict endocrine sensitivity and survival in ER+ BC patients.

A CTC-Cluster-Specific Signature Derived from OMICS Analysis of Patient-Derived Xenograft Tumors Predicts Outcomes in Basal-Like Breast Cancer.

Circulating tumor cell clusters (CTCcl) have a higher metastatic potential compared to single CTCs and predict long-term outcomes in breast cancer (BC) patients. Because of the rarity of CTCcls, molecular characterization of primary tumors that give rise to CTCcl hold significant promise for better diagnosis and target discovery to combat metastatic BC. In our study, we utilized the reverse-phase protein array (RPPA) and transcriptomic (RNA-Seq) data of 10 triple-negative BC patient-derived xenograft (TNBC PDX) transplantable models with CTCs and evaluated expression of upregulated candidate protein Bcl2 (B-cell lymphoma 2) by immunohistochemistry (IHC). The sample-set consisted of six CTCcl-negative (CTCcl-) and four CTCcl-positive (CTCcl+) models. We analyzed the RPPA and transcriptomic profiles of CTCcl- and CTCcl+ TNBC PDX models. In addition, we derived a CTCcl-specific gene signature for testing if it predicted outcomes using a publicly available dataset from 360 patients with basal-like BC. The RPPA analysis of CTCcl+ vs. CTCcl- TNBC PDX tumors revealed elevated expression of Bcl2 (false discovery rate (FDR) < 0.0001, fold change (FC) = 3.5) and reduced acetyl coenzyme A carboxylase-1 (ACC1) (FDR = 0.0005, FC = 0.3) in CTCcl+ compared to CTCcl- tumors. Genome-wide transcriptomic analysis of CTCcl+ vs. CTCcl- tumors revealed 549 differentially expressed genes associated with the presence of CTCcls. Apoptosis was one of the significantly downregulated pathways (normalized enrichment score (NES) = -1.69; FDR < 0.05) in TNBC PDX tumors associated with CTCcl positivity. Two out of four CTCcl+ TNBC PDX primary tumors had high Bcl2 expression by IHC (H-score > 34); whereas, only one of six CTCcl- TNBC PDX primary tumors met this criterion. Evaluation of epithelial-mesenchymal transition (EMT)-specific signature did not show significant differences between CTCcl+ and CTCcl- tumors. However, a gene signature associated with the presence of CTCcls in TNBC PDX models was associated with worse relapse-free survival in the publicly available dataset from 360 patients with basal-like BC. In summary, we identified the multigene signature of primary PDX tumors associated with the presence of CTCcls. Evaluation of additional TNBC PDX models and patients can further illuminate cellular and molecular pathways facilitating CTCcl formation.

Novel SUMO-Protease SENP7S Regulates ß-catenin Signaling and Mammary Epithelial Cell Transformation.

SUMO post-translational modification of proteins or SUMOylation ensures normal cell function. Disruption of SUMO dynamics prompts various pathophysiological conditions, including cancer. The burden of deSUMOylating the large SUMO-proteome rests on 6 full-length mammalian SUMO-proteases or SENP. While multiple SENP isoforms exist, the function of these isoforms remains undefined. We now delineate the biological role of a novel SENP7 isoform SENP7S in mammary epithelial cells. SENP7S is the predominant SENP transcript in human mammary epithelia but is significantly reduced in precancerous ductal carcinoma in situ and all breast cancer subtypes. Like other SENP family members, SENP7S has SUMO isopeptidase activity but unlike full-length SENP7L, SENP7S is localized in the cytosol. In vivo, SUMOylated ß-catenin and Axin1 are both SENP7S-substrates. With knockdown of SENP7S in mammary epithelial cells, Axin1-ß-catenin interaction is lost and ß-catenin escapes ubiquitylation-dependent proteasomal degradation. SUMOylated ß-catenin accumulates at the chromatin and activates multiple oncogenes. Hence, non-tumorigenic MCF10-2A cells with reduced SENP7S exhibit greater cell proliferation and anchorage-dependent growth. SENP7S depletion directly potentiates tumorigenic properties of MCF10-2A cells with induction of anchorage-independent growth and self-renewal in 3D-spheroid conditions. Collectively, the results identify SENP7S as a novel mediator of ß-catenin signaling and normal mammary epithelial cell physiology.

A major allergen in rainbow trout (Oncorhynchus mykiss): complete sequences of parvalbumin by MALDI tandem mass spectrometry.

Fish parvalbumin (PRVB) is an abundant and stable protein in fish meat. The variation in cross-reactivity among individuals is well known and explained by a broad repertoire of molecular forms and differences between IgE-binding epitopes in fish species. PVRB has "sequential" epitopes, which retain their IgE-binding capacity and allergenicity also after heating and digestion using proteolytic enzymes. From the allergonomics perspective, PRVB is still a challenging target due to its multiple isoforms present at different degrees of distribution. Little information is available in the databases about PVRBs from Oncorhynchus mykiss. At present, only two validated, incomplete isoforms of this species are included in the protein databases: parvalbumin beta 1 (P86431) and parvalbumin beta 2 (P86432). A simple and rapid protocol has been developed for selective solubilization of PRVB from the muscle of farmed rainbow trout (Oncorhynchus mykiss), followed by calcium depletion, proteolytic digestion, MALDI MS, and MS/MS analysis. With this strategy thermal allergen release was assessed and PRVB1 (P86431), PRVB1.1, PRVB2 (P86432) and PRVB2.1 variants from the rainbow trout were sequenced. The correct ordering of peptide sequences was aided by mapping the overlapping enzymatic digests. The deduced peptide sequences were arranged and the theoretical molecular masses (Mr) of the resulting sequences were calculated. Experimental masses (Mr) of each PRVB variant were measured by linear MALDI-TOF.

Mass spectrometry and potentiometry studies of Pb(II)-, Cd(II)- and Zn(II)-cystine complexes.

Cd(II)-, Pb(II)- and Zn(II)-cystine complexes were investigated by potentiometric and different mass spectrometric (MS) methodologies. Laser desorption mass spectrometry has provided both the composition and structure of metal-cystine complexes according to the speciation models proposed on the basis of the potentiometric data. Detection of neutral complexes was achieved by protonation or electrochemical reduction during mass spectrometric experiments. The redox activity of metal-cystine complexes was confirmed by laser desorption and charge transfer matrix assisted laser assisted MS experiments, which allowed us to observe the formation of complexes with a reduction of cystine. The stoichiometry of Cd(II)-, Pb(II)- and Zn(II)-cystine complexes was defined by observing the isotopic pattern of the investigated compound. The results suggest that interaction occurs through the carboxylate group of the ligand.