Biology subjects

Barinka, C.

Publications and source records attributed to Barinka, C..

9 recordsLinked to original sources

bioRxiv2026

HDAC6 and SSAT2 orchestrate acetyllysine metabolism and protein homeostasis

Lysine acetylation is a prevalent and dynamic posttranslational modification, yet the metabolic fate and physiological role of free N {varepsilon}-acetyl-L-lysine (AcK) remain unclear. Here, we identify opposing enzyme-catalyzed reactions linking lysine/AcK homeostasis to protein stability. We show that the first catalytic domain of HDAC6 stereospecifically deacetylates free AcK, establishing HDAC6 as an N {varepsilon}-acyl-lysine deacylase. AcK functions both as a recyclable lysine reservoir and as a signaling metabolite that elevates -tubulin acetylation and suppresses cell migration. Conversely, SSAT2 acetylates free lysine in cells and undergoes substrate-dependent C-terminal autoacetylation. We demonstrate that C-terminal lysine acetylation constitutes a reversible proteasomal degradation signal that promotes SSAT2 degradation and may extend to additional proteins bearing a terminal lysine. Stabilization of SSAT2 by HDAC6-dependent deacetylation, together with intricate regulation affected by substrate availability, reveals a novel mechanism that couples amino acid recycling to cytoskeletal dynamics and proteostasis.

biochemistry↗

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

Accurate and fast segmentation of filaments and membranes in micrographs and tomograms with TARDIS

Segmentation of macromolecular structures is the primary bottleneck for studying biomolecules and their organization with electron microscopy in 2D/3D - requiring months of manual effort. Transformer-based Rapid Dimensionless Instance Segmentation (TARDIS) is a deep learning framework that automatically and accurately annotates membranes and filaments. Pre-trained TARDIS models can segment electron tomography (ET) reconstructions from both 3D and 2D electron micrographs of cryo and plastic-embedded samples. Furthermore, by implementing a novel geometric transformer architecture, TARDIS is the only method to provide accurate instance segmentations of these structures. Reducing the annotation time for ET data from months to minutes, we demonstrate segmentation of membranes and filaments in over 13,000 tomograms in the CZII Data Portal. TARDIS thus enables quantitative biophysical analysis at scale for the first time. We show this in application to kinetochore-microtubule attachment and viral-membrane interactions. TARDIS can be extended to new biomolecules and applications and open-source at https://github.com/SMLC-NYSBC/TARDIS.

bioinformatics↗

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↗

bioRxiv2023

A new mechanism of posttranslational polyglutamylation regulates phase separation and signaling of the Wnt pathway protein Dishevelled.

Polyglutamylation is a reversible post-translational modification that is catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Disheveled 3 (DVL3), thereby changing the interactome of DVL3, as well as it increases its capacity to get phosphorylated, to undergo liquid-liquid phase separation (LLPS), and to act in the non-canonical Wnt pathway. Both carboxyterminal polyglutamylation and the resulting reduction in LLPS capacity of DVL3 were reverted by the deglutamylating enzyme CCP6, which demonstrates the causal relationship between TTLL11-mediated polyglutamylation and LLPS. We thus discovered a novel type of posttranslational modification, which significantly broadens the range of proteins that can be modified by polyglutamylation and provides the first evidence that polyglutamylation can act as a regulator of protein LLPS.

cell biology↗

bioRxiv2023

Uncovering the essential roles of human GCP 2 orthologs in Caenorhabditis elegans.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. This study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to human GCP2 physiology and in extension to GCP2 involvement in pathophysiological processes.

biochemistry↗

bioRxiv2020

Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential processes including neuronal activity. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under protein crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in crowded intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases its robustness of stepping in protein crowding conditions. Interaction with TRAK1 i) facilitated kinesin-1 navigation around obstacles, ii) increased the probability of kinesin-1 passing through cohesive envelopes of tau and iii) increased the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.

cell 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↗