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 (FcγRI/CD64) on the surface of phagocytes. Functional parts of the 5D3-CP33 engager revealed a nanomolar affinity for PSMA and FcγRI/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 the 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 0.15 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 developing new immunotherapy tools for the efficient treatment of PCa.

• MeSH
• Antigens, Surface * immunology   metabolism   MeSH
• Glutamate Carboxypeptidase II * immunology   metabolism   MeSH
• Immunotherapy methods   MeSH
• Humans MeSH
• Macrophages immunology   MeSH
• Monocytes * immunology   metabolism   MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms * immunology   therapy   pathology   MeSH
• Antibodies, Bispecific * immunology   pharmacology   MeSH
• Receptors, IgG metabolism   immunology   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Surface * MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II * MeSH
• Antibodies, Bispecific * MeSH
• Receptors, IgG MeSH

Retinitis pigmentosa (RP) is a hereditary disorder caused by mutations in more than 70 different genes including those that encode proteins important for pre-mRNA splicing. Most RP-associated mutations in splicing factors reduce either their expression, stability or incorporation into functional splicing complexes. However, we have previously shown that two RP mutations in PRPF8 (F2314L and Y2334N) and two in SNRNP200 (S1087L and R1090L) behaved differently, and it was still unclear how these mutations affect the functions of both proteins. To investigate this in the context of functional spliceosomes, we used iCLIP in HeLa and retinal pigment epithelial (RPE) cells. We found that both mutations in the RNA helicase SNRNP200 change its interaction with U4 and U6 snRNAs. The significantly broader binding profile of mutated SNRNP200 within the U4 region upstream of the U4/U6 stem I strongly suggests that its activity to unwind snRNAs is impaired. This was confirmed by FRAP measurements and helicase activity assays comparing mutant and WT protein. The RP variants of PRPF8 did not affect snRNAs, but showed a reduced binding to pre-mRNAs, which resulted in the slower splicing of introns and altered expression of hundreds of genes in RPE cells. This suggests that changes in the expression and splicing of specific genes are the main driver of retinal degeneration in PRPF8-linked RP.

• Keywords
• PRPF8, Pre-mRNA splicing, Retinitis pigmentosa, SNRNP200, iCLIP,
• MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   genetics   MeSH
• Mutation * MeSH
• RNA Precursors * metabolism   genetics   MeSH
• RNA-Binding Proteins * metabolism   genetics   MeSH
• Retinal Pigment Epithelium metabolism   MeSH
• Retinitis Pigmentosa * genetics   metabolism   pathology   MeSH
• Ribonucleoproteins, Small Nuclear * metabolism   genetics   MeSH
• RNA, Small Nuclear * metabolism   genetics   MeSH
• RNA Splicing genetics   MeSH
• RNA Splicing Factors metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ribonucleoprotein, U4-U6 Small Nuclear MeSH
• RNA Precursors * MeSH
• RNA-Binding Proteins * MeSH
• PRPF8 protein, human MeSH Browser
• Ribonucleoproteins, Small Nuclear * MeSH
• RNA, Small Nuclear * MeSH
• RNA Splicing Factors MeSH
• SNRNP200 protein, human MeSH Browser

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is 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 Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

• Keywords
• Dishevelled 3, Noncanonical Wnt Signaling, Polyglutamylation, Protein Condensates, TTLL11,
• MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Polyglutamic Acid metabolism   analogs & derivatives   MeSH
• Humans MeSH
• Peptide Synthases * metabolism   genetics   MeSH
• Protein Processing, Post-Translational * MeSH
• Dishevelled Proteins * metabolism   genetics   MeSH
• Phase Separation MeSH
• Wnt Signaling Pathway MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DVL3 protein, human MeSH Browser
• Polyglutamic Acid MeSH
• Peptide Synthases * MeSH
• Dishevelled Proteins * MeSH
• tubulin polyglutamylase MeSH Browser

Histone deacetylase (HDAC) inhibitors used in the clinic typically contain a hydroxamate zinc-binding group (ZBG). However, more recent work has shown that the use of alternative ZBGs, and, in particular, the heterocyclic oxadiazoles, can confer higher isoenzyme selectivity and more favorable ADMET profiles. Herein, we report on the synthesis and biochemical, crystallographic, and computational characterization of a series of oxadiazole-based inhibitors selectively targeting the HDAC6 isoform. Surprisingly, but in line with a very recent finding reported in the literature, a crystal structure of the HDAC6/inhibitor complex revealed that hydrolysis of the oxadiazole ring transforms the parent oxadiazole into an acylhydrazide through a sequence of two hydrolytic steps. An identical cleavage pattern was also observed both in vitro using the purified HDAC6 enzyme as well as in cellular systems. By employing advanced quantum and molecular mechanics (QM/MM) and QM calculations, we elucidated the mechanistic details of the two hydrolytic steps to obtain a comprehensive mechanistic view of the double hydrolysis of the oxadiazole ring. This was achieved by fully characterizing the reaction coordinate, including identification of the structures of all intermediates and transition states, together with calculations of their respective activation (free) energies. In addition, we ruled out several (intuitively) competing pathways. The computed data (ΔG‡ ≈ 21 kcal·mol-1 for the rate-determining step of the overall dual hydrolysis) are in very good agreement with the experimentally determined rate constants, which a posteriori supports the proposed reaction mechanism. We also clearly (and quantitatively) explain the role of the -CF3 or -CHF2 substituent on the oxadiazole ring, which is a prerequisite for hydrolysis to occur. Overall, our data provide compelling evidence that the oxadiazole warheads can be efficiently transformed within the active sites of target metallohydrolases to afford reaction products possessing distinct selectivity and inhibition profiles.

• MeSH
• Histone Deacetylase 6 chemistry   MeSH
• Hydrolysis MeSH
• Histone Deacetylase Inhibitors * pharmacology   MeSH
• Hydroxamic Acids chemistry   MeSH
• Oxadiazoles * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histone Deacetylase 6 MeSH
• Histone Deacetylase Inhibitors * MeSH
• Hydroxamic Acids MeSH
• Oxadiazoles * MeSH

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

• Keywords
• histone deacetylase, inhibitor profiling, metallohydrolase, nanoBRET, tubulin/histone acetylation,
• MeSH
• Acetylation MeSH
• Histone Deacetylase 6 * antagonists & inhibitors   MeSH
• Histone Deacetylases * metabolism   MeSH
• Histone Deacetylase Inhibitors * chemistry   pharmacology   MeSH
• Hydroxamic Acids * chemistry   pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Oxadiazoles * chemistry   pharmacology   MeSH
• Protein Processing, Post-Translational MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• HDAC10 protein, human MeSH Browser
• Histone Deacetylase 6 * MeSH
• Histone Deacetylases * MeSH
• Histone Deacetylase Inhibitors * MeSH
• Hydroxamic Acids * MeSH
• Oxadiazoles * MeSH

INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

• Keywords
• N-glycosylation, NAALADase I, PSMA, folate hydrolase, glutamate carboxypeptidase II, site-specific glycoform,
• MeSH
• Antigens, Surface metabolism   MeSH
• Cell Line MeSH
• Glutamate Carboxypeptidase II metabolism   MeSH
• Glycosylation MeSH
• Mass Spectrometry methods   MeSH
• Humans MeSH
• Biomarkers, Tumor analysis   MeSH
• Prostatic Neoplasms * metabolism   pathology   MeSH
• Polysaccharides * classification   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Prostate * enzymology   metabolism   pathology   MeSH
• Neoplasm Staging MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Biomarkers, Tumor MeSH
• Polysaccharides * MeSH

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

• MeSH
• Cell Line MeSH
• Escherichia coli genetics   MeSH
• Flavoproteins biosynthesis   genetics   isolation & purification   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mitochondrial Proteins biosynthesis   genetics   isolation & purification   MeSH
• Protoporphyrinogen Oxidase biosynthesis   genetics   isolation & purification   MeSH
• Recombinant Fusion Proteins biosynthesis   genetics   isolation & purification   MeSH
• Sf9 Cells MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Flavoproteins MeSH
• Mitochondrial Proteins MeSH
• PPOX protein, human MeSH Browser
• Protoporphyrinogen Oxidase MeSH
• Recombinant Fusion Proteins MeSH

Extracellular signal-regulated kinase (ERK) is a part of the mitogen-activated protein kinase (MAPK) signaling pathway which allows the transduction of various cellular signals to final effectors and regulation of elementary cellular processes. Deregulation of the MAPK signaling occurs under many pathological conditions including neurodegenerative disorders, metabolic syndromes and cancers. Targeted inhibition of individual kinases of the MAPK signaling pathway using synthetic compounds represents a promising way to effective anti-cancer therapy. Cross-talk of the MAPK signaling pathway with other proteins and signaling pathways have a crucial impact on clinical outcomes of targeted therapies and plays important role during development of drug resistance in cancers. We discuss cross-talk of the MAPK/ERK signaling pathway with other signaling pathways, in particular interplay with the Hippo/MST pathway. We demonstrate the mechanism of cell death induction shared between MAPK/ERK and Hippo/MST signaling pathways and discuss the potential of combination targeting of these pathways in the development of more effective anti-cancer therapies.

• Keywords
• ERK, Hippo, MAPK, MST, PI3K, YAP, apoptosis, cancer, caspase, inhibitors, natural compounds, therapy,
• MeSH
• Extracellular Signal-Regulated MAP Kinases antagonists & inhibitors   metabolism   MeSH
• Protein Kinase Inhibitors therapeutic use   MeSH
• Humans MeSH
• Neoplasms drug therapy   metabolism   MeSH
• Protein Serine-Threonine Kinases antagonists & inhibitors   metabolism   MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Hippo Signaling Pathway MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Extracellular Signal-Regulated MAP Kinases MeSH
• Protein Kinase Inhibitors MeSH
• Protein Serine-Threonine Kinases MeSH
• Antineoplastic Agents MeSH

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.

• Keywords
• cytoskeleton, histone deacetylase 6 (HDAC6), intrinsically disordered protein, microtubule-associated protein (MAP), post-translational modification, protein motif, protein-protein interaction, structure-function, substrate specificity, total internal reflection fluorescence (TIRF), tubulin,
• MeSH
• Acetylation MeSH
• Histone Deacetylase 6 metabolism   MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Protein Domains physiology   MeSH
• Amino Acid Sequence MeSH
• Substrate Specificity MeSH
• Tubulin metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• HDAC6 protein, human MeSH Browser
• Histone Deacetylase 6 MeSH
• Tubulin MeSH