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Formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimerʼs disease (AD). Here, we present the three-dimensional structure of an Aβ oligomer formed in a membrane mimicking environment, namely an Aβ(1-42) tetramer, which comprises a six stranded β-sheet core. The two faces of the β-sheet core are hydrophobic and surrounded by the membrane-mimicking environment while the edges are hydrophilic and solvent-exposed. By increasing the concentration of Aβ(1-42) in the sample, Aβ(1-42) octamers are also formed, made by two Aβ(1-42) tetramers facing each other forming a β-sandwich structure. Notably, Aβ(1-42) tetramers and octamers inserted into lipid bilayers as well-defined pores. To establish oligomer structure-membrane activity relationships, molecular dynamics simulations were carried out. These studies revealed a mechanism of membrane disruption in which water permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core β-sheets edges of the oligomers. Formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimer´s disease. Here authors present the 3D- structure of an Aβ oligomer formed in a membrane mimicking environment and observe that Aβ tetramers and octamers inserted into lipid bilayers as well-defined pores.

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 mechanism of iron-sulfur (Fe-S) cluster biosynthesis is not fully understood. Here, the authors develop a physiologically relevant in vitro model of Fe-S cluster assembly, allowing them to elucidate the sequence of Fe-S cluster synthesis along with the respective roles of ferredoxin-2 and frataxin.

TRIM24 (TIF1α), TRIM28 (TIF1β), and TRIM33 (TIF1γ) are three related cofactors belonging to the tripartite motif superfamily that interact with distinct transcription factors. TRIM24 interacts with the liganded retinoic acid (RA) receptor to repress its transcriptional activity. Germ line inactivation of TRIM24 in mice deregulates RA-signaling in hepatocytes leading to the development of hepatocellular carcinoma (HCC). Here we show that TRIM24 can be purified as at least two macromolecular complexes comprising either TRIM33 or TRIM33 and TRIM28. Somatic hepatocyte-specific inactivation of TRIM24, TRIM28, or TRIM33 all promote HCC in a cell-autonomous manner in mice. Moreover, HCC formation upon TRIM24 inactivation is strongly potentiated by further loss of TRIM33. These results demonstrate that the TIF1-related subfamily of TRIM proteins interact both physically and functionally to modulate HCC formation in mice.

Antibody-drug conjugates (ADCs) are highly complex proteins mainly due to the structural microvariability of the mAb, along with the additional heterogeneity afforded by the bioconjugation process. Top-down (TD) and middle-down (MD) strategies allow the straightforward fragmentation of proteins to elucidate the conjugated amino acid residues. Nevertheless, these spectra are very crowded with multiple overlapping and unassigned ion fragments. Here we report on the use of dedicated software (ClipsMS) and application of proton transfer charge reduction (PTCR), to respectively expand the fragment ion search space to internal fragments and improve the separation of overlapping fragment ions for a more comprehensive characterization of a recently approved ADC, trastuzumab deruxtecan (T-DXd). Subunit fragmentation allowed between 70 and 90% of sequence coverage to be obtained. Upon addition of internal fragment assignment, the three subunits were fully sequenced, although internal fragments did not contribute significantly to the localization of the payloads. Finally, the use of PTCR after subunit fragmentation provided a moderate sequence coverage increase between 2 and 13%. The reaction efficiently decluttered the fragmentation spectra allowing increasing the number of fragment ions characteristic of the conjugation site by 1.5- to 2.5-fold. Altogether, these results show the interest in the implementation of internal fragment ion searches and more particularly the use of PTCR reactions to increase the number of signature ions to elucidate the conjugation sites and enhance the overall sequence coverage of ADCs, making this approach particularly appealing for its implementation in R&D laboratories.

Many physiological processes are dependent on G protein-coupled receptors (GPCRs), the biggest family of human membrane proteins and a significant class of therapeutic targets. Once activated by external stimuli, GPCRs use G proteins and arrestins as transducers to generate second messengers and trigger downstream signaling, leading to diverse signaling profiles. The G protein-coupled bile acid receptor 1 (GPBAR1, also known as Takeda G protein-coupled receptor 5, TGR5) is a class A bile acid membrane receptor that regulates energy homeostasis and glucose and lipid metabolism. GPBAR1/Gs protein interactions are implicated in the prevention of diabetes and the reduction of inflammatory responses, making GPBAR1 a potential therapeutic target for metabolic disorders. Here, we present combined hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) to identify the molecular determinants of GPBAR1 conformational dynamics upon G protein binding. Thanks to extensive optimization, we achieved over 75% sequence coverage by HDX-MS of a complete GPCR complex and a 2.5 Å resolution structure by cryo-EM, both of which are state-of-the-art. Altogether, our results provide information on the under-investigated GPBAR1 binding mode to its cognate G protein, pinpointing the synergic and powerful combination of higher cryo-EM and lower HDX-MS resolution techniques to dissect GPCR/G protein binding characteristics.

Here we present the synthesis and evaluation of antibody-drug conjugates (ADCs), for which antibody and drug are non-covalently connected using complementary DNA linkers. These ADCs are composed of trastuzumab, an antibody targeting HER2 receptors overexpressed on breast cancer cells, and monomethyl auristatin E (MMAE) as a drug payload. In this new ADC format, trastuzumab conjugated to a 37-mer oligonucleotide (ON) was prepared and hybridized with its complementary ON modified at 5-end with MMAE (cON-MMAE) in order to obtain trastuzumab-DNA-MMAE. As an advantage, the cON-MMAE was completely soluble in water, which decreases overall hydrophobicity of toxic payload, an important characteristic of ADCs. The stability in the human plasma of these non-engineered ON-based linkers was investigated and showed a satisfactory half-life of 5.8 days for the trastuzumab-DNA format. Finally, we investigated the in vitro cytotoxicity profile of both the DNA-linked ADC and the ON-drug conjugates and compared them with classical covalently linked ADC. Interestingly, we found increased cytotoxicity for MMAE compared to cON-MMAE and an EC50 in the nanomolar range for trastuzumab-DNA-MMAE on HER2-positive cells. Although this proved to be less potent than classically linked ADC with picomolar range EC50, the difference in cytotoxicity between naked payload and conjugated payload was significant when an ON linker was used. We also observed an interesting increase in cytotoxicity of trastuzumab-DNA-MMAE on HER2-negative cells. This was attributed to enhanced non-specific interaction triggered by the DNA strand as it could be confirmed using ligand tracer assay.

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC–MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC–MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC–MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC–MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC–MS platforms for high-throughput determination of major CQAs in a regulated environment.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25D3), regulates a number of physiological and pathological processes, including cell proliferation and differentiation. Thousands of analogues of 1,25D3 have been developed with the aim of selective effects for medical use. Here we describe the synthesis of two new unconventional vitamin D analogues bearing A-ring modifications with ortho-carborane (dicarba-o-closo-1,2-dodecaborane) units. The ligands function as agonists for VDR with similar antiproliferative activities as 1,25D3. Whereas mice treated with the analogues 4 and 5 exhibited similar hypercalcemic activities as 1,25D3, only compound 4 and 1,25D3 induced the strong activation of CYP24A1 mRNA expression but not compound 5.

Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention. Zika virus (ZIKV) can infect the central nervous system, but it is not clear how it reaches the brain. Here, Ayala-Nunez et al. show in ex vivo and in vivo models that ZIKV can hitch a ride in monocytes in a Trojan Horse manner to cross the endothelium and disseminate the virus.

Antibody-Oligonucleotide Conjugates (AOCs) represent an emerging class of functionalized antibodies that have already been used in a wide variety of applications. While the impact of dye and drug conjugation on antibodies’ ability to bind their target has been extensively studied, little is known about the effect caused by the conjugation of hydrophilic and charged payloads such as oligonucleotides on the functions of an antibody. Previous observations of non-specific interactions of nucleic acids with untargeted cells prompted us to further investigate their impact on AOC binding abilities and cell selectivity. We synthesized a series of single- and double-stranded AOCs, as well as a human serum albumin-oligonucleotide conjugate, and studied their interactions with both targeted and non-targeted living cells using a time-resolved analysis of ligand binding assay. Our results indicate that conjugation of single strand oligonucleotides to proteins induce consistent non-specific interactions with cell surfaces while double strand oligonucleotides have little or no effect, depending on the preparation method.