A Discussion with Dr. Natasha Kekre, Hematologist and Clinician Scientist.

[Figure: see text] Dr. Natasha Kekre has been appointed to the Department of Medicine in the Division of Hematology, within the Transplant and Cellular Therapy Program at The Ottawa Hospital since 2015. She is also a scientist within the Ottawa Hospital Research Institute and an associate professor of medicine at the University of Ottawa. She completed her Bachelor's in Science at the University of Windsor then obtained her medical degree from the University of Ottawa. She trained at the University of Ottawa in Internal Medicine and Hematology, then did fellowship in stem cell transplantation at Dana Farber Cancer Institute in Boston, MA with a Masters in Public Health from Harvard University. Her research is focused on developing early phase clinical trials and moving home grown therapeutic strategies from the lab to patients in the clinic. She has collaborated with scientists and physicians across Canada to build a Canadian CAR-T cell platform (chimeric antigen receptor T cells are immune cells engineered to kill cancer cells), bringing this exciting new therapy to Canadian patients.

Supermetalation of Cd-MT3 beyond the two-domain model.

The flexibility of mammalian metallothioneins (MTs) has contributed to the difficulty in obtaining structural information for this family of metalloproteins that bind divalent metals with its twenty cysteines. While the two-domain structure for Cd7MT is well-established as a Cd4S11 and Cd3S9, a third structure has been reported when 8 Cd(II) ions bind to MT1. Isoform 3 of the MT family, MT3, has been of interest to the research community since its isolation as a growth inhibitory factor isolated in brain tissue, and has since been noted as a prominent participant in the mediation of neurodegenerative diseases and regular brain development. The differences between MT3 and the other isoforms of MT include an additional hexapeptide insertion of acidic residues in the α domain as well as the introduction of two prolines in the ß domain. It is unclear whether these changes impact the metalation properties of MT3. We report the formation of a Cd8MT3 species is characterized by electrospray ionization mass spectrometry and UV-visible absorption spectroscopy. We report that the spectroscopic properties of this supermetalated Cd8MT3 are similar to those of the supermetalated Cd8MT1, with a clear indication of changes in structure from "fully-metalated" Cd7MT3 to supermetalated Cd8MT3 from circular dichroism spectra and both 1D 113Cd and 2D 1H-113Cd HSQC NMR spectra. We conclude that the metalation properties are not impacted significantly due to the amino acid changes in MT3, and that the cysteinyl thiols are the key players in determining the capacity of metal-binding and the structure of metal-thiolate clusters.

Metallothionein-3 and carbonic anhydrase metalation properties with Zn(II) and Cd(II) change as a result of protein-protein interactions.

Metallothioneins (MT) are regulators of the metals Zn(II) and Cu(I) and act as antioxidants in many organisms, including in humans. Isoform 3 (MT3) is expressed constitutively in central nervous tissue and has been shown to have additional biological functions, including the inhibition of neuronal growth, the regulation of apoptosis, and cytoskeleton modulation. To facilitate these functions, protein-protein interactions likely occur. These interactions may then impact the metalation status of the MT and the recipient metalloprotein. Using electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report that the interaction between the zinc metalloenzyme, carbonic anhydrase (CA), and MT3, impacts the metalation profiles of both apo-MT3 and apo-CA with Cd(II) and Zn(II). We observe two phases in the metalation of the apo-CA, the first of which is associated with an increased binding affinity of apo-CA for Cd/Zn(II) and the second pathway is associated with apo-CA metalated without a change in binding affinity. The weak interactions that result in this change of binding affinity are not detectable as a protein complex in the ESI-mass spectral data or in the circular dichroism spectra. These unusual metalation properties of apo-CA in the presence of apo-MT3 are evidence of the effects of protein-protein interactions. With adjustment to take into account the interaction of both proteins, we report the complete Cd(II) and Zn(II) binding constants of MT3 under physiological conditions, as well as the pH dependence of these binding pathways.

Overview of The the Clinician Investigator Trainees' Research Presented at The 2022 CSCI-CITAC Joint Meeting.

The 2022 Annual Joint Meeting (AJM) and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de recherches clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada/Association des cliniciens-chercheurs en formation du Canada (CITAC/ACCFC) was held in Montréal, November 13-14, 2022. The theme of this year's AJM was "Strength in Perseverance" and focused on highlighting clinician-investigator trainee achievements and resilience in research engagement through recent challenging and unprecedented times. The opening remarks were given by Nicola Jones (president of CSCI/SCRC) and Heather Whittaker (past president of CITAC/ACCFC). The keynote speaker was Dr. Michael Strong, who delivered the presentation "The Future of Clinician Scientists in Canada." Dr. Caroline Quach (Université de Montréal) received the CSCI Distinguished Scientist Award and Dr. Amy Metcalfe (University of Calgary) received the CSCI Joe Doupe Young Investigator Award. Each of the clinician-scientists delivered presentations on their award-winning research. The four interactive workshops included "Social Media in Science and Medicine," "Diversity in Science and Medicine," "Running a Successful Research Program," and "Mentorship in Action." The AJM also included presentations from clinician investigator trainees from across the country. Over 90 abstracts were showcased at this year's meeting, most of which are summarized in this review. Six outstanding abstracts were selected for oral presentations during the President's Forum.

Fall 2023: Clinician Investigator Trainee Association of Canada (CITAC).

This has been a great first half of the year for CITAC-ACCFC (Clinician Investigator Trainee Association of Canada/Association des cliniciens-chercheurs en formation du Canada)! We are looking forward to our new members joining us in the fall and welcoming back our previous members after the summer.

Xenobiotic Bi3+ Coordination by Cysteine-Rich Metallothionein-3 Reveals a Cooperatively Formed Thiolate-Sharing Bi2S5 Cluster.

The field of designing artificial metalloproteins has yet to effectively tackle the incorporation of multimetal clusters, which is a key component of natural metalloproteins, such as metallothioneins (MTs) and calmodulin. MT is a physiological, essential, cysteine-rich metalloprotein that binds to a variety of metals but is only known to form metal-thiolate clusters with Cd2+, Zn2+, and Cu+. Bismuth is a xenobiotic metal and a component of metallodrugs used to treat gastric ulcers and cancer, as well as an emerging metal used in industrial practices. Electrospray ionization mass spectrometry, UV-visible spectroscopy, and extended X-ray absorption fine structure spectroscopy were used to probe the Bi3+ binding site structures in apo-MT3 (brain-located MT) at pH 7.4 and 2 and provide the complete set of binding affinities. We discovered the highly cooperative formation of a novel Bi3+ species, Bi2MT3, under physiological conditions, where each Bi3+ ion is coordinated by three cysteinyl thiolates, with one of the thiolates bridging between the two Bi3+ ions. This cluster structure was associated with a strong visible region absorption band, which was disrupted by the addition of Zn2+ and reversibly disrupted by acidification and increased temperature. This is the first reported presence of bridging cysteines for a xenobiotic metal in MT3 and the Bi2MT structure is the first Bi cluster found in a metalloprotein.

Arsenic binding to human metallothionein-3.

Arsenic poisoning is of great concern with respect to its neurological toxicity, which is especially significant for young children. Human exposure to arsenic occurs worldwide from contaminated drinking water. In human physiology, one response to toxic metals is through coordination with the metallochaperone metallothionein (MT). Central nervous system expression of MT isoform 3 (MT3) is thought to be neuroprotective. We report for the first time on the metalation pathways of As3+ binding to apo-MT3 under physiological conditions, yielding the absolute binding constants (log Kn, n = 1-6) for each sequential As3+ binding event: 10.20, 10.02, 9.79, 9.48, 9.06, and 8.31 M-1. We report on the rate of the reaction of As3+ with apo-MT3 at pH 3.5 with rate constants (kn, n = 1-6) determined for each sequential As3+ binding event: 116.9, 101.2, 85.6, 64.0, 43.9, and 21.0 M-1 s-1. We further characterize the As3+ binding pathway to fully metalated Zn7MT3 and partially metalated Zn-MT3. As3+ binds rapidly with high binding constants under physiological conditions in a noncooperative manner, but is unable to replace the Zn2+ in fully-metalated Zn-MT3. As3+ binding to partially metalated Zn-MT3 takes place with a rearrangement of the Zn-binding profile. Our work shows that As 3+ rapidly and efficiently binds to both apo-MT3 and partially metalated Zn-MT3 at physiological pH.

Apo-metallothionein-3 cooperatively forms tightly compact structures under physiological conditions.

Metallothioneins (MTs) are essential mammalian metal chaperones. MT isoform 1 (MT1) is expressed in the kidneys and isoform 3 (MT3) is expressed in nervous tissue. For MTs, the solution-based NMR structure was determined for metal-bound MT1 and MT2, and only one X-ray diffraction structure on a crystallized mixed metal-bound MT2 has been reported. The structure of solution-based metalated MT3 is partially known using NMR methods; however, little is known about the fluxional de novo apo-MT3 because the structure cannot be determined by traditional methods. Here, we used cysteine modification coupled with electrospray ionization mass spectrometry, denaturing reactions with guanidinium chloride, stopped-flow methods measuring cysteine modification and metalation, and ion mobility mass spectrometry to reveal that apo-MT3 adopts a compact structure under physiological conditions and an extended structure under denaturing conditions, with no intermediates. Compared with apo-MT1, we found that this compact apo-MT3 binds to a cysteine modifier more cooperatively at equilibrium and 0.5 times the rate, providing quantitative evidence that many of the 20 cysteines of apo-MT3 are less accessible than those of apo-MT1. In addition, this compact apo-MT3 can be identified as a distinct population using ion mobility mass spectrometry. Furthermore, proposed structural models can be calculated using molecular dynamics methods. Collectively, these findings provide support for MT3 acting as a noninducible regulator of the nervous system compared with MT1 as an inducible scavenger of trace metals and toxic metals in the kidneys.

Spring 2022: Clinician Investigator Trainee Association Of Canada (CITAC).

Over the past two years, physician-scientist trainees have persevered in the face of evolving challenges presented by the ongoing coronavirus disease 2019 (COVID-19) pandemic. Research and healthcare institutions across the country continue to feel the impacts of the public health emergency. As scientists and physicians generate evidence to inform the prevention and treatment of COVID-19, physician-scientist trainees in all disciplines have adapted to the changing conditions of their education.

Interplay between Carbonic Anhydrases and Metallothioneins: Structural Control of Metalation.

Carbonic anhydrases (CAs) and metallothioneins (MTs) are both families of zinc metalloproteins central to life, however, they coordinate and interact with their Zn2+ ion cofactors in completely different ways. CAs and MTs are highly sensitive to the cellular environment and play key roles in maintaining cellular homeostasis. In addition, CAs and MTs have multiple isoforms with differentiated regulation. This review discusses current literature regarding these two families of metalloproteins in carcinogenesis, with a dialogue on the association of these two ubiquitous proteins in vitro in the context of metalation. Metalation of CA by Zn-MT and Cd-MT is described. Evidence for protein-protein interactions is introduced from changes in metalation profiles of MT from electrospray ionization mass spectrometry and the metalation rate from stopped-flow kinetics. The implications on cellular control of pH and metal donation is also discussed in the context of diseased states.

Metallothionein Cd4S11 cluster formation dominates in the protection of carbonic anhydrase.

Metallothioneins (MTs) are ubiquitous proteins vital for essential metal homeostasis and heavy metal detoxification. The twenty-cysteinyl mammalian metallothioneins protect the proteome by sequestering heavy metals into thermodynamically stable metal thiolate structures when metalated with seven Cd2+. At physiological pH, the first metal (M) thiolate (SCys) structures formed involve M(SCys)4 terminal thiolates. With higher metal loading, M4(SCys)11 and M3(SCys)9 clusters form. As a regulator of the metallome, it is necessary to understand metal sequestration properties of MT in solution with other metalloproteins. We report that the association between apo-MT and apo-carbonic anhydrase (CA) enhances the formation of the protective mode of MT, in which Cd4(SCys)11-clusters form at much lower concentration levels than for the free apo-MT at physiological pH. Using stopped-flow kinetics and electrospray ionization mass spectrometry, we quantified this protective effect, determining that it is significant at pH 7.4, but the effect diminishes at pH 5.0. We report for the first time, the absolute stepwise binding constants of Cd2+ binding to human MT1a both in the presence and absence of CA through calibration by the known binding constant of Cd2+ to bovine CA. We report that this protein association affects the Cd2+ metalation rates of MT. The data support the physiological role of MTs as protectors of the metalloproteome from the toxic effects of Cd2+.