Biology subjects

Novakova, Z.

Publications and source records attributed to Novakova, Z..

4 recordsLinked to original sources

bioRxiv2025

Expanding the Tubulin Code: TTLL11 Polyglutamylase Drives Elongation of Primary Tubulin Chains

Microtubules (MTs) undergo diverse post-translational modifications that regulate their structural and functional properties. Among these, polyglutamylation - a dominant and conserved modification targeting the unstructured tubulin C-terminal tails - plays a pivotal role in defining the tubulin code. Here, we uncovered a novel mechanism by which tubulin tyrosine ligase-like 11 (TTLL11) expands and diversifies the code. Cryo-electron microscopy revealed a unique bipartite MT recognition strategy wherein TTLL11s binding and catalytic domains engage adjacent MT protofilaments. Biochemical assays identified previously unknown polyglutamylation patterns, showing that TTLL11 directly extends the primary polypeptide chains of - and {beta}-tubulin, challenging the prevailing paradigms emphasizing lateral branching. Moreover, cell-based and in vivo data firmly established a crosstalk between TTLL11-mediated polyglutamylation and other tubulin-modifying processes, notably the detyrosination/tyrosination cycle. This discovery unveils an unrecognized layer of complexity within the tubulin code and offers new insights into the molecular basis of functional specialization of cytoskeleton across diverse cellular contexts.

biochemistry↗

bioRxiv2024

Targeting prostate cancer by new bispecific monocyte engager directed to prostate-specific membrane antigen

Prostate cancer (PCa) ranks as the second leading cause of cancer-related deaths among men in the United States. Prostate-specific membrane antigen (PSMA) represents a well-established biomarker of PCa and its levels correlate positively with the disease progression, culminating at the stage of metastatic castration-resistant prostate cancer. Due to its tissue-specific expression and cell surface localization, PSMA shows superior potential for precise imaging and therapy of PCa. Antibody-based immunotherapy targeting PSMA offers the promise of selectively engaging the host immune system with minimal off-target effects. Here we report on the design, expression, purification, and characterization of a bispecific engager, termed 5D3-CP33, that efficiently recruits macrophages to the vicinity of PSMA-positive cancer cells mediating PCa death. The engager was engineered by fusing the anti-PSMA 5D3 antibody fragment to a cyclic peptide 33 (CP33) selectively binding the Fc gamma receptor I (Fc{gamma}RI/CD64) on the surface of phagocytes. Functional parts of 5D3-CP33 engager revealed nanomolar affinity for PSMA and Fc{gamma}RI/CD64 with dissociation constants of KD = 3 nM and KD = 140 nM, respectively. At a concentration as low as 0.3 nM, the engager was found to trigger production of reactive oxygen species by U937 monocytic cells in the presence of PSMA-positive cells. Moreover, flow cytometry analysis demonstrated antibody-dependent cell-mediated phagocytosis of PSMA-positive cancer cells by U937 monocytes when exposed to 0.1 nM 5D3-CP33. Our findings illustrate that 5D3-CP33 effectively and specifically activates monocytes upon PSMA-positive target engagement, resulting in the elimination of tumor cells. The 5D3-CP33 engager can thus serve as a promising lead for the development of new immunotherapy tools for the efficient treatment of PCa.

bioengineering↗

bioRxiv2023

Galectin-3-binding protein inhibits extracellular heparan 6-O-endosulfatse Sulf-2

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). In spite of its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the secretome of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP has functional relevance, and may regulate physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

cancer biology↗

bioRxiv2017

HDAC11 is a fatty-acid deacylase

Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides exceed 70,000 M-1s-1 making HDAC11 the most proficient fatty acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic and stearic acids with inhibition constants of 6.5 {micro}M, 0.9 {micro}M, and 1.6 {micro}M, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by fatty acid metabolites. Our data reveal novel enzymatic activity of HDAC11 that can, in turn, facilitate the uncovering of additional biological functions of the enzyme as well as the design of isoform-specific HDAC inhibitors.

biochemistry↗