Not all eplet mismatches are created equal - A cohort study illustrating implications to long-term graft outcomes.

We assessed implications of various eplet-compatibility strategies to death-censored graft failure (DCGF), defined as return to dialysis or re-transplantation, in a base-case scenario from the Scientific Registry of Transplant Recipients. To inform personalized care, we evaluated how recipient, donor, and transplant characteristics affect DCGF by ascending categories of eplet mismatches (EMM), and derived adjusted hazard ratios (HR). The base-case analysis demonstrated 15-year estimated survival probabilities of 77.1%, 75.4%, 73.6%, 72.2%, 74.9%, and 73.5% for the lowest EMM categories (complete epitype: 0-19, antibody-verified (AbVer) epitype and class II eplets: 0-9, class II AbVer eplets: 0-4, 55 high-risk eplets associated with DCGF: 0-3, and subset of 15 high-risk eplets validated in an independent subcohort: 0 EMM, respectively). Beyond the lowest EMM categories, the Epi15 strategy allowed better differentiation of change in DCGF risk per EMM, with additional 5.2%, 3.9% and 4.1% decrease in estimated graft survival for each additional EMM (1, 2, and ≥ 3, respectively). Recipients < 25 years, donors > 55 years, and immunosuppression regimens excluding calcineurin inhibitors and steroids, demonstrated higher HR for DCGF. High-risk EMM allowed better differentiation between DCGF probabilities per EMM, suggesting that recipients at higher risk for graft failure could benefit most from allocation schemes ensuring compatibility on these eplets.

Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I.

BACKGROUND: Cardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI135-209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI146-158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI1-37, regulates the calcium affinity of cTnC. cTnI is known to be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in ischemia-reperfusion injury. METHODS: Soluble fragments of cTnI containing its N- and C-terminal tails, cTnI1-77 and cTnI135-209, were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both constructs using liquid chromatography-mass spectrometry and solution NMR studies of the C-terminal tail. RESULTS: cTnI135-209 is intrinsically disordered, though it contains three regions with helical propensity (including the switch region) that acquire more structure upon actin binding. We identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical cTnI switch region is targeted by both proteases. CONCLUSIONS: Both N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be understood in terms of their interactions as probed by NMR studies. GENERAL SIGNIFICANCE: cTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use as gold standard biomarker of myocardial injury.

Proteolytic Digestion of Serum Cardiac Troponin I as Marker of Ischemic Severity.

BACKGROUND: The serum troponin assay is the biochemical gold standard for detecting myocardial infarction (MI). A major diagnostic issue is that some believe troponin levels can rise with reversible injury, in the absence of radiologically detectable infarct. HYPOTHESIS: Because cell death activates intracellular proteases, troponin released by irreversible infarct will be more proteolyzed than that released by milder processes. Our goal was to quantify proteolytic digestion of cardiac troponin I in patients with varying degrees of myocardial injury. METHODS: Serum or plasma samples from 29 patients with cardiac troponin I elevations were analyzed for proteolytic degradation, using 3 different sandwich ELISAs designed to specifically detect the N-terminal, core, or C-terminal regions of cardiac troponin I. RESULTS: As predicted, the degree of proteolytic digestion increased with increasing severity of injury, as estimated by the total troponin level, and this trend was more pronounced for C-terminal (vs N-terminal) degradation. The highest degree of proteolytic digestion was observed in patients with ST-elevation MI; the least, in type 2 MI (supply-demand ischemia rather than acute thrombus formation). CONCLUSIONS: The proteolytic degradation pattern of cardiac troponin I may be a better indicator of clinically significant MI than total serum troponin level. Distinguishing between intact and degraded forms of troponin may be useful for (a) identifying those patients with clinically significant infarct in need of revascularization, (b) monitoring intracellular proteolysis as a possible target for therapeutic intervention, and (c) providing an impetus for standardizing the epitopes used in the troponin I assay.

Combining a PagP fusion protein system with nickel ion-catalyzed cleavage to produce intrinsically disordered proteins in E. coli.

Many proteins contain intrinsically disordered regions that are highly solvent-exposed and susceptible to post-translational modifications. Studying these protein segments is critical to understanding their physiologic regulation, but proteolytic degradation can make them difficult to express and purify. We have designed a new protein expression vector that fuses the target protein to the N-terminus of the integral membrane protein, PagP. The two proteins are connected by a short linker containing the sequence SRHW, previously shown to be optimal for nickel ion-catalyzed cleavage. The methodology is demonstrated for an intrinsically disordered segment of cardiac troponin I. cTnI[135-209]-SRHW-PagP-His6 fusion protein was overexpressed in Escherichia coli, accumulating in insoluble inclusion bodies. The protein was solubilized, purified using nickel affinity chromatography, and then cleaved with 0.5mM NiSO4 at pH 9.0 and 45 °C, all in 6M guanidine-HCl. Nickel ion-catalyzed peptide bond hydrolysis is an effective chemical cleavage technique under denaturing conditions that preclude the use of proteases. Moreover, nickel-catalyzed cleavage is more specific than the most commonly used agent, cyanogen bromide, which cleaves C-terminal to methionine residues. We were able to produce 15 mg of purified cTnI[135-209] from 1L of M9 minimal media using this protocol. The methodology is more generally applicable to the production of intrinsically disordered protein segments.