A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth.

Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome.

Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends.

Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, are stable and grow very slowly, but the underlying mechanisms are poorly understood. Here, we reconstituted in vitro the interplay between the proteins that cap distal centriole ends and control their elongation: CP110, CEP97, and CPAP/SAS-4.

Affinity-based covalent sialyltransferase probes enabled by ligand-directed chemistry.

Sialyltransferases (ST) are key enzymes found in, among others, mammals and bacteria that are responsible for producing sialylated glycans, which play critical roles in human health and disease. However, chemical tools to study sialyltransferases have been limited to non-covalent inhibitors and probes that do not allow isolation and profiling of these important enzymes. Here we report a new class of covalent affinity-based probes (AfBP) for ST by using ligand-directed chemistry (LDchem).

Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags.

Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets.

Variable Assembly and Procapsid Binding of Bacteriophage P22 Terminase Subunits in Solution.

Concatemeric viral DNA is packaged into bacteriophage P22 procapsids via a headful packaging mechanism mediated by a molecular machine consisting of small (gp3) and large (gp2) terminase subunits. Although a negative stain reconstruction exists for the terminase holoenzyme, it is not clear how this complex binds the dodecameric portal protein located at a 5-fold mismatch vertex. Herein, we describe new assemblies for the holoenzyme.

Multi-tiered proteome analysis displays the hyper-permeability of the rheumatoid synovial compartment for plasma proteins.

Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and cartilage/bone destruction. RA affects the synovial joints, the synovial lining and the permeability of the synovium. As the latter is of central relevance for the distribution of systemically delivered therapeutics into synovial fluid (SF), we here assessed the protein composition of paired plasma and SF of patients diagnosed with RA at three distinct levels of depth using mass spectrometric approaches: the "total" proteome, the "total" IgG1 antibody repertoire and the RA-specific ACPA IgG1 autoantibody repertoire.

π-HuB: the proteomic navigator of the human body.

The human body contains trillions of cells, classified into specific cell types, with diverse morphologies and functions. In addition, cells of the same type can assume different states within an individual's body during their lifetime. Understanding the complexities of the proteome in the context of a human organism and its many potential states is a necessary requirement to understanding human biology, but these complexities can neither be predicted from the genome, nor have they been systematically measurable with available technologies.

Human IgG Subclasses Differ in the Structural Elements of Their -Glycosylation.

Although immunoglobulin G (IgG) harbors just one -glycosylation site per heavy chain, this glycosylation plays a key role in modulating its function. In human serum, IgG is classified into four subclasses (IgG1, IgG2, IgG3, IgG4), each characterized by unique features in their sequences, disulfide bridges and glycosylation signatures. While protein glycosylation is typically studied at the compositional level, this severely underestimates the complexity of the molecules involved.

Integrated transcriptomics- and structure-based drug repositioning identifies drugs with proteasome inhibitor properties.

Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells.

LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.

Probing recombinant AAV capsid integrity and genome release after thermal stress by mass photometry.

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated () through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs.

Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed Drugs.

Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-target interactions, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of the drug selectivity and efficacy.

A neutralizing antibody prevents postfusion transition of measles virus fusion protein.

Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use.

Higher-order structure and proteoforms of co-occurring C4b-binding protein assemblies in human serum.

The complement is a conserved cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP).

Proteomic dissection of vanishing white matter pathogenesis.

Vanishing white matter (VWM) is a leukodystrophy caused by biallelic pathogenic variants in eukaryotic translation initiation factor 2B. To date, it remains unclear which factors contribute to VWM pathogenesis. Here, we investigated the basis of VWM pathogenesis using the 2b5 mouse model.

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS.

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging.

Region-specific and age-related differences in astrocytes in the human brain.

Astrocyte heterogeneity and its relation to aging in the normal human brain remain poorly understood. We here analyzed astrocytes in gray and white matter brain tissues obtained from donors ranging in age between the neonatal period to over 100 years. We show that astrocytes are differently distributed with higher density in the white matter.

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce.

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant.

Using proteomics and complexome profiling, we evaluated in a year-long study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2-glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14.

Protein-Centric Analysis of Personalized Antibody Repertoires Using LC-MS-Based Fab-Profiling on a timsTOF.

Endogenous antibodies, or immunoglobulins (Igs), abundantly present in body fluids, represent some of the most challenging samples to analyze, largely due to the immense variability in their sequences and concentrations. It has been estimated that our body can produce billions of different Ig proteins with different isotypes, making their individual analysis seemingly impossible. However, recent advances in protein-centric proteomics using LC-MS coupled to Orbitrap mass analyzers to profile intact Fab fragments formed by selective cleavage at the IgG-hinge revealed that IgG repertoires may be less diverse, albeit unique for each donor.

Antigen-specific Fab profiling achieves molecular-resolution analysis of human autoantibody repertoires in rheumatoid arthritis.

The presence of autoantibodies is a defining feature of many autoimmune diseases. The number of unique autoantibody clones is conceivably limited by immune tolerance mechanisms, but unknown due to limitations of the currently applied technologies. Here, we introduce an autoantigen-specific liquid chromatography-mass spectrometry-based IgG1 Fab profiling approach using the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA) as an example.

Assessment of Kinome-Wide Activity Remodeling upon Picornavirus Infection.

Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g.

Lipidome characterisation and sex-specific differences in type 1 and type 2 diabetes mellitus.

Background: In this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state.

Methods: An untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative).

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC.

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components.