Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

High-Resolution IMS-MS to Assign Additional Disulfide Bridge Pairing in Complementarity-Determining Regions of an IgG4 Monoclonal Antibody.

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the complementarity-determining regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb-collected and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs.

Investigating Ugi/Passerini Multicomponent Reactions for the Site-Selective Conjugation of Native Trastuzumab*.

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the great majority of existing methods that solely target a single residue. We opted for the Ugi four-centre three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads; furthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.

VHH characterization.Recombinant VHHs: Production, characterization and affinity.

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology.

VHH characterization. Comparison of recombinant with chemically synthesized anti-HER2 VHH.

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates.

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin.

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.

The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein.

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, we demonstrated the involvement of mNT in a specific cytosolic pathway dedicated to the reactivation of oxidatively damaged cytosolic aconitase by cluster transfer. In vitro studies using apo-ferredoxin (FDX) reveal that mNT uses an Fe-based redox switch mechanism to regulate the transfer of its cluster. Using the "gold standard" cluster recipient protein, FDX, we show that this transfer is direct and that only one of the two mNT clusters is transferred when the second one is decomposed. Combining complementary biophysical and biochemical approaches, we show that pH affects both the sensitivity of the cluster to O2 and dimer stability. Around physiological cytosolic pH, the ability of mNT to transfer its cluster is tightly regulated by the pH. Finally, mNT is extremely resistant to H2O2 compared to ISCU and SufB, two other Fe-S cluster transfer proteins, which is consistent with its involvement in a repair pathway of stress-damaged Fe-S proteins. Taken together, our results suggest that the ability of mNT to transfer its cluster to recipient proteins is not only controlled by the redox state of its cluster but also tightly modulated by the pH of the cytosol. We propose that when pathophysiological conditions such as cancer and neurodegenerative diseases dysregulate cellular pH homeostasis, this pH-dependent regulation of mNT is lost, as is the regulation of cellular pathways under the control of mNT.

Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates.

Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies.