Biology subjects

Kadava, T.

Publications and source records attributed to Kadava, T..

6 recordsLinked to original sources

bioRxiv2026

Mycoplasma immunoglobulin binding protein universally binds human antibodies, thereby reducing Fab arm flexibility and displacing antigens in immune complexes

Mycoplasma immunoglobulin binding (MIB) protein plays a key role in immune evasion across several Mycoplasma species. MIB tightly interacts with the Fab domain of immunoglobulin G, inducing conformational changes that disrupt the antigen-binding site. Here, we reveal that MIB binds strongly and universally to all major isotypes and subclasses of human antibodies and can thus serve as a bait protein to efficiently deplete antibodies from serum. Our results demonstrate strong interactions between MIB and a diverse set of both recombinant monoclonal and endogenous polyclonal antibodies, the latter purified from serum or colostrum. All antibodies, including IgG and IgA monomers, J-chain coupled IgA dimers, and IgM pentamers, consistently bind two copies of MIB per antibody protomer. Using cross-linking mass spectrometry, we pinpoint that antibody-MIB interactions involve conserved regions of the variable and constant antibody domains, independent of isotype. Furthermore, we demonstrate that MIB not only disrupts the Fab-antigen interaction but also restricts Fab flexibility, as visualized by atomic force microscopy. Conversely, MIB does not affect Fc-mediated protein interactions, as exemplified by the successful reconstitution of a 36-subunit immune complex, (IgG)6-(MIB)12-C1q.

biochemistry↗

bioRxiv2025

Uncovering the link between ATP synthase and the TCA cycle by crosslinking mass spectrometry

Mitochondrial oxidative phosphorylation (OXPHOS) comprises a series of multi-subunit protein complexes that operate in coordination with the tricarboxylic acid (TCA) cycle to generate ATP. Although these systems are metabolically interconnected, complex II is generally regarded as the only direct structural link between the OXPHOS and TCA cycle. Here, we combine in-solution crosslinking mass-spectrometry (XL-MS), quantitative proteomics, and blue native PAGE (BN-PAGE) to explore how ATP synthase (complex V) integrates within the mitochondrial metabolic network under physiological and pathological conditions. We demonstrate that in murine wild-type hearts, the F1 catalytic head of ATP synthase forms extensive contacts with TCA cycle enzymes, establishing a previously unanticipated link between the OXPHOS and central carbon metabolism. We also found that under mitochondrial dysfunction, in this case LRPPRC-deficient hearts, where defective mitochondrial DNA gene expression destabilizes ATP synthase, these interactions become strengthened. Moreover, ATP synthase dysfunction promotes binding of the ATPase inhibitory factor 1 (ATIF1) to the F1 head via its N-terminal inhibitory region, shifting the ATP synthase toward an energy-preserving state. Together, our findings show that ATP synthase deficiency drives remodeling of the F1 interactome, revealing how mitochondrial structure and regulation adapt to preserve energy homeostasis under stress.

biochemistry↗

bioRxiv2025

Deep Coverage and Extended Sequence Reads Obtained with a Single Archaeal Protease Expedite de novo Protein Sequencing by Mass Spectrometry

The ability to sequence proteins without reliance on a genomic template defines a critical frontier in modern proteomics. This approach, known as de novo protein sequencing, is essential for applications such as antibody sequencing, microbiome proteomics, and antigen discovery, which require accurate reconstruction of peptide and protein sequences. While trypsin remains the gold-standard protease in proteomics, its restricted cleavage specificity limits peptide diversity. This constraint is especially problematic in antibody sequencing, where the functionally critical regions often lack canonical tryptic sites. As a result, exclusively trypsin-based approaches yield sparse reads, leading to sequence gaps. Multi-protease and hybrid-fragmentation strategies can improve the sequence coverage, but they add complexity, compromise scalability and reproducibility. Here, we explore two HyperThermoacidic Archaeal (HTA)-proteases as single-enzyme solutions for de novo antibody sequencing. Each HTA-protease generated about five times more unique peptide reads than trypsin or chymotrypsin, providing high redundancy across all CDRs. Combined with EAciD fragmentation on a ZenoTOF 7600 system, this approach enabled complete, unambiguous antibody sequencing. De novo analysis using PEAKS/DeepNovo and Stitch showed up to fourfold higher alignment scores and reduced the sequence errors within the HTA-generated data. Additionally, the HTA-EAciD approach offers short digestion times, eliminates extensive cleanup, and enables analysis in a single LC-MS/MS run. This streamlined, single-protease approach delivers therefore performance comparable to multi-enzyme workflows, offering a scalable and efficient strategy for de novo protein sequencing across diverse applications.

biochemistry↗

bioRxiv2024

Intra-manchette transport employs both microtubule and actin tracks

The manchette is a transient microtubule based structure that plays a vital role in nuclear shaping during spermio-genesis. It comprises thousands of microtubules (MTs) that build a scaffold around the distal half of the nucleus. The manchette distributes proteins and vesicles during spermio-genesis in a process called intra-manchette transport (IMT). The current hypothesis is that IMT shares many similarities with intra-flagellar transport (IFT) and utilizes both MTs and filamentous actin (F-actin). However, IMT is still poorly understood as direct visualization of IMT complexes is missing, and the presence of F-actin has not been experimentally shown. Here, we use proteomics and cryogenic-electron tomography (cryo-ET) to identify and visualize IMT components. We find that F-actin is an integral part of the manchette with two different spatial organizations, namely bundles and single filaments, providing tracks for transport as well as having structural and mechanical roles. We further uncover that IMT on MTs is mediated by two distinct transport machineries: a dynein-mediated transport of soluble cargo and a dynein-independent transport for vesicles. Our results provide new insights into the manchettes function as a transport scaffold, highlighting its significance for the polarization of spermatids during spermiogenesis.

cell biology↗

bioRxiv2024

Bispecific antibodies against the hepatitis C virus E1E2 envelope glycoprotein

Hepatitis C virus (HCV) currently causes about one million infections and 240,000 deaths worldwide each year. To reach the goal set by the World Health Organization (WHO) of global HCV elimination by 2030, it is critical to develop a prophylactic vaccine. Broadly neutralizing antibodies (bNAbs) target the E1E2 envelope glycoproteins on the viral surface, can neutralize a broad range of the highly diverse circulating HCV strains and are essential tools to inform vaccine design. However, bNAbs targeting a single E1E2 epitope might be limited in neutralization breadth, which can be enhanced by using combinations of bNAbs that target different envelope epitopes. We have generated 60 IgG-like bispecific antibodies (bsAbs) that can simultaneously target two distinct epitopes on E1E2. We combine non-overlapping E1E2 specificities into three types of bsAbs, each containing a different hinge length. The bsAbs show retained or increased potency and breadth against a diverse panel of HCV pseudoparticles (HCVpp) and HCV produced in cell culture (HCVcc) compared to monospecific and cocktail controls. Additionally, we demonstrate that changes in the hinge length of bsAbs can alter the binding stoichiometry to E1E2. These results provide insights into the binding modes and the role of avidity in bivalent targeting of diverse E1E2 epitopes, and suggest structural differences between HCVpp and HCVcc. This study illustrates how potential cooperative effects of HCV bNAbs can be utilized by strategically designing bispecific constructs. These new HCV bsAbs can guide vaccine development and unlock novel therapeutic and prophylactic strategies against HCV and other (flavi)viruses.

microbiology↗

bioRxiv2021

In vitro evolution reveals primordial RNA-protein interaction mediated by metal cations

RNA-peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA-protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal L11 C-terminal domain was selected from a [~]1010 library of partially randomized sequences, all composed of 10 prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with MD simulations uncover qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant L11 domain uses bridging ion (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein-RNA interactions evolved, where the lack of aromatic/basic residues was compensated by acidic residues plus metal ions.

biochemistry↗