a Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M 4 S 11 (a) and M 3 S 9 (b) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano-to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resolution with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the a-domain is formed sequentially in the first stages, followed by the formation of the b-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS 4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the b-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(II) ion associates with the b-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated b-domain differs significantly from the whole protein, which explains its previously observed different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated b-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis. Significance to metallomicsThe growing interest in metallomics within metal-dependent biological systems provides an opportunity to explore the structural and buffering properties of metallothioneins (MTs). The wide relevance of MTs is obvious due to their multiple biological functions such as participation in zinc and copper homeostasis. However, the MT metalloproteome is still not fully explored and is rather complicated in vivo or in vitro because of its highly dynamic structure, which lacks secondary structural elements and metamorphic behaviour. We have undertaken compreh...

Aging is a multifactorial process characterized by the progressive loss of physiological functions, leading to an increased vulnerability to age-associated diseases and finally to death. Several theories have been proposed to explain the nature of aging. One of the most known identifies the free radicals produced by the mitochondrial metabolism as the cause of cellular and DNA damage. However, there are also several evidences supporting that epigenetic modifications, such as DNA methylation, noncoding RNAs, and histone modifications, play a critical role in the molecular mechanism of aging. In this review, we explore the significance of these findings and argue how the interlinked effects of oxidative stress and epigenetics can explain the cause of age-related declines.

Non-small cell lung cancer (NSCLC) is a subtype of the most frequently diagnosed cancer in the world. Its epidemiology depends not only on tobacco exposition but also air quality. While the global trends in NSCLC incidence have started to decline, we can observe region-dependent differences related to the education and the economic level of the patients. Due to an increasing understanding of NSCLC biology, new diagnostic and therapeutic strategies have been developed, such as the reorganization of histopathological classification or tumor genotyping. Precision medicine is focused on the recognition of a genetic mutation in lung cancer cells called “driver mutation” to provide a variety of specific inhibitors of improperly functioning proteins. A rapidly growing group of approved drugs for targeted therapy in NSCLC currently allows the following mutated proteins to be treated: EGFR family (ERBB-1, ERBB-2), ALK, ROS1, MET, RET, NTRK, and RAF. Nevertheless, one of the most frequent NSCLC molecular sub-types remains without successful treatment: the K-Ras protein. In this review, we discuss the current NSCLC landscape treatment focusing on targeted therapy and immunotherapy, including first- and second-line monotherapies, immune checkpoint inhibitors with chemotherapy treatment, and approved predictive biomarkers.

Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two α- and β-domains and represent a major cellular Zn­(II)/Cu­(I) buffering system in the cell. At cellular free Zn­(II) concentrations (10–11–10–9 M), MTs do not exist in fully loaded forms with seven Zn­(II)-bound ions (Zn7MTs). Instead, MTs exist as partially metal-depleted species (Zn4–6MT) because their Zn­(II) binding affinities are on the nano- to picomolar range comparable to the concentrations of cellular Zn­(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn­(II) (un)­binding to MTs, the thermodynamic properties of the Zn1–6MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quantitative bottom-up and top-down MS in combination with steered molecular dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5 μs) were integrated to unravel the chemical coordination of Zn­(II) in all Zn1–6MT2 species and to explain the differences in binding affinities of Zn­(II) ions to MTs. Differences are found to be the result of the degree of water participation in MT (un)­folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodynamics properties of Zn­(II) (un)­binding to MT2 are found to differ from those of Cd­(II), justifying their distinctive roles. The potential of this integrated strategy in the investigation of numerous unexplored metalloproteins is attested by the results highlighted in the present study.

Metallothionein and glutathione are key players of the fate of Cu(ii)–/Zn(ii)–/Fe(ii)–thiosemicarbazone anticancer drugs in the cytosol/nucleus.

Here, using human metallothionein (MT2) as an example, we describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity utilizing iodoacetamide (IAM) and N -ethylmaleimide reagents. We concluded that an S N 2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissociation. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addition reaction after separation by reverse-phase C18 at acidic pH. Finally, we evaluated the efficiency of the method by mapping metal-binding sites of Zn 7– x MT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resolution. The methodology presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-containing proteins.

Silver (Ag(i)) binding to consensus zinc fingers (ZFs) causes Zn(ii) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgnSn clusters.

Copper complexes are of medicinal and biological interest, including as anticancer drugs designed to cleave intracellular biomolecules by O 2 activation. To exhibit such activity,the copper complex must be redox active and resistant to dissociation. Metallothioneins (MTs) and glutathione (GSH) are abundant in the cytosol and nucleus.Because they are thiol-richr educing molecules with high Cu I affinity,t hey are potential competitors for ac opper ion bound in ac opper drug. Herein, we report the investigation of apanel of Cu I /Cu II complexes often used as drugs,w ith diverse coordination chemistries and redox potentials.W ee valuated their catalytic activity in ascorbate oxidation based on redox cycling between Cu I and Cu II ,a sw ell as their resistance to dissociation or inactivation under cytosolically relevant concentrations of GSH and MT.O 2 -activating Cu I /Cu II complexes for cytosolic/nuclear targets are generally not stable against the GSH/MT system, whichcreates achallenge for their future design.