Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Soluble B-cell maturation antigen in lacrimal fluid as a potential biomarker and mediator of keratopathy in multiple myeloma.

Belantamab mafodotin (belantamab) is a first-in-class anti-BCMA antibody-drug conjugate approved for the treatment of triple-class refractory multiple myeloma. It provides a unique therapeutic option for patients ineligible for CAR-T and bispecific antibody therapy, and/or patients progressing on anti-CD38 treatment where CAR-T and bispecifics might be kept in reserve. Wider use of the drug can be challenged by its distinct ocular side effect profile, including corneal microcysts and keratopathy. While dose reduction has been the most effective way to reduce these toxicities, the underlying mechanism of this BCMA off-target effect remains to be characterized. In this study, we provide the first evidence for soluble BCMA (sBCMA) in lacrimal fluid and report on its correlation with tumor burden in myeloma patients. We confirm that corneal cells do not express BCMA, and show that sBCMA-belantamab complexes may rather be internalized by corneal epithelial cells through receptor-ligand independent pinocytosis. Using an hTcEpi corneal cell-line model, we show that the pinocytosis inhibitor EIPA significantly reduces belantamab-specific cell killing. As a proof of concept, we provide detailed patient profiles demonstrating that, after belantamab-induced cell killing, sBCMA is released into circulation, followed by a delayed increase of sBCMA in the tear fluid and subsequent onset of keratopathy. Based on the proposed mechanism, pinocytosis-induced keratopathy can be prevented by lowering the entry of sBCMA into the lacrimal fluid. Future therapeutic concepts may therefore consist of belantamab-free debulking therapy prior to belantamab consolidation and/or concomitant use of gamma-secretase inhibition as currently evaluated for belantamab and nirogacestat in ongoing studies.

Combination of Betulinic Acid Fragments and Carbonic Anhydrase Inhibitors-A New Drug Targeting Approach.

Human carbonic anhydrase IX (hCA IX) is a zinc(II)-dependent metalloenzyme that plays a critical role in the conversion of carbon dioxide and water to protons and bicarbonate. It is a membrane-bound protein with an extracellular catalytic center that is predominantly overexpressed in solid hypoxic tumors. Sulfamates and sulfonamides, for example acetazolamide (AZA), have been used to inhibit hCA IX in order to improve the response to solid hypoxic tumors. In the present study, we propose a new drug targeting approach by attaching the natural cytotoxic substances betulin and betulinic acid (BA) via a linker to sulfonamides. The conjugate was designed with different spacer lengths to accumulate at the target site of hCA IX. Computational and cell biological studies suggest that the length of the linker may influence hCA IX inhibition. Cytotoxicity tests of the newly synthesized bifunctional conjugates 3, 5, and 9 show effective cytotoxicity in the range of 6.4 and 30.1 µM in 2D and 3D tumor models. The hCA IX inhibition constants of this conjugates, measured using an in vitro enzyme assay with p-nitrophenyl acetate, were determined in a low µM-range, and all compounds reveal a significant inhibition of hypoxia-induced CA activity in a cell-based assay using the Wilbur-Anderson method. In addition, the cells respond with G1 increase and apoptosis induction. Overall, the dual strategy to produce cytotoxic tumor therapeutics that inhibit tumor-associated hCA IX was successfully implemented.

Long-Term Outcome of Corneal Collagen Crosslinking with Riboflavin and UV-A Irradiation for Keratoconus.

PURPOSE: To evaluate long-term outcomes of corneal collagen crosslinking (CXL) using riboflavin and UV-A irradiation and to determine when to repeat CXL. METHODS: In this retrospective consecutive interventional case series 131 eyes of 131 patients (95 male, 36 female, mean age 29.7 ± 11.4 years) between 2006 and 2016 received standard CXL (Dresden protocol, epithelium-off) for progressive keratoconus. Corrected distance visual acuity (CDVA) and corneal tomography (K1, K2, Kmax) were repeatedly recorded 1 year (n = 103 eyes) to 10 years (n = 44) postoperatively. Only one eye per patient was included. Paired t-test or Wilcoxon matched-pairs signed rank test was used for parametric and nonparametric data, respectively. RESULTS: 1-3 years preoperatively, median K2 significantly increased by 1.1 D (p < 0.001). Postoperatively, median K2 increased by 0.1 D after 1 year, then decreased over the remaining postoperative period by 0.85 D (p = 0.021). Kmax fluctuated without significant change. Median apical corneal thickness decreased by 16 µm (p = 0.012) after 5 years and then returned to preoperative values. Mean CDVA showed a significant improvement (decrease in logMAR 0.08 after 10 years, p = 0.010). CXL non-responders, defined by a postoperative increase in Kmax>2 D, increased from 16% after 5 to 33% after 10 years. Risk factors for non-response were young age, high astigmatism (>4.3 D), thin cornea (<480 µm), poor initial visual acuity (CDVA ≥0.3 D), and atopic dermatitis. 4 eyes were re-treated 3-4 years after first CXL without complications and keratoconus stabilized thereafter. CONCLUSIONS: CXL can slow or stop keratoconus progression. However, as the number of responders declines after 5 years, especially patients with risk factors may require re-treatment.

Crucial Role of the Chaperonin GroES/EL for Heterologous Production of the Soluble Methane Monooxygenase from Methylomonas methanica MC09.

Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C-H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co-synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non-heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.

CD28 Superagonist D665-mediated activation of mouse regulatory T cells maintains their phenotype without loss of suppressive quality.

Regulatory T cells (Tregs) maintain immune homeostasis by regulating the activation of other immune cells. Preclinical studies show that the infusion of Tregs can promote immunological tolerance to allografts and prevent or cure multiple autoimmune diseases. However, Treg therapy is limited by high numbers of cells required to induce tolerance. In this study, we aimed at improving the in vitro expansion of sort purified mouse Tregs using the CD28 Superagonist (CD28-SA) D665 and comparing it to the conventional expansion using anti-CD3/anti-CD28 Dynabeads®. CD28-SA-stimulated Tregs expanded more than Dynabead®-stimulated Tregs while maintaining their phenotype by expressing the same level of CD4, CD25 and Foxp3. CD28-SA-expanded Tregs produced comparable amounts of IL-10 and TGFß while showing a slightly superior suppressive capacity compared to Dynabead®-stimulated Tregs. Thus, stimulating murine Tregs with the CD28-SA is a promising alternative since it maintains their suppressive capacity without altering their phenotype and yields a higher fold expansion within 14 days.

Cardiolipin Stabilizes and Increases Catalytic Efficiency of Carnitine Palmitoyltransferase II and Its Variants S113L, P50H, and Y479F.

Muscle carnitine palmitoyltransferase II (CPT II) deficiency is associated with various mutations in CPT2 gene. In the present study, the impact of the two CPT II variants P50H and Y479F were characterized in terms of stability and activity in vitro in comparison to wildtype (WT) and the well investigated variant S113L. While the initial enzyme activity of all variants showed wild-type-like behavior, the activity half-lives of the variants at different temperatures were severely reduced. This finding was validated by the investigation of thermostability of the enzymes using nano differential scanning fluorimetry (nanoDSF). Further, it was studied whether the protein stabilizing diphosphatidylglycerol cardiolipin (CL) has an effect on the variants. CL indeed had a positive effect on the stability. This effect was strongest for WT and least pronounced for variant P50H. Additionally, CL improved the catalytic efficiency for CPT II WT and the investigated variants by twofold when carnitine was the varied substrate due to a decrease in KM. However, there was no influence detected for the variation of substrate palmitoyl-CoA. The functional consequences of the stabilization by CL in vivo remain open.

Hydrazides Are Potent Transition-State Analogues for Glutaminyl Cyclase Implicated in the Pathogenesis of Alzheimer's Disease.

Amyloidogenic plaques are hallmarks of Alzheimer's disease (AD) and typically consist of high percentages of modified Aß peptides bearing N-terminally cyclized glutamate residues. The human zinc(II) enzyme glutaminyl cyclase (QC) was shown in vivo to catalyze the cyclization of N-terminal glutamates of Aß peptides in a pathophysiological side reaction establishing QC as a druggable target for therapeutic treatment of AD. Here, we report crystallographic snapshots of human QC catalysis acting on the neurohormone neurotensin that delineate the stereochemical course of catalysis and suggest that hydrazides could mimic the transition state of peptide cyclization and deamidation. This hypothesis is validated by a sparse-matrix inhibitor screening campaign that identifies hydrazides as the most potent metal-binding group compared to classic Zn binders. The structural basis of hydrazide inhibition is illuminated by X-ray structure analysis of human QC in complex with a hydrazide-bearing peptide inhibitor and reveals a pentacoordinated Zn complex. Our findings inform novel strategies in the design of potent and highly selective QC inhibitors by employing hydrazides as the metal-binding warhead.

Synthesis and biological investigation of new carbonic anhydrase IX (CAIX) inhibitors.

In this report, we describe the synthesis, characterization, in vitro anticancer activity and Carbonic anhydrase IX (CAIX) inhibition of new sulfamate conjugates of Betulin and Betulinic acid (BA). The betulinyl sulfamates were subjected to inhibit carbonic anhydrases (CA), e.g. CAIX, an attractive target for tumor-selective therapy strategies in cancer cells. Data on combined in vitro antitumor activity with CAIX inhibition are very rare. The betulinyl sulfamates were tested against five tumor cell lines and normal human skin fibroblasts. The mode of cell death on MCF7 breast cancer cells induced by the most active compounds CAI1, CAI3 and CAI6 was investigated by Fluorescence Activated Cell Sorting (FACS) experiments. The compounds showed inhibitory activity towards CAIX, which was determined via in vitro enz-yme assay. Our preliminary investigations revealed that all compounds showed potent anticancer properties with IC50 values below 20 µM against all tumor cells. Interestingly, among the panel of sulfamate conjugates, CAI3 found to be highly cytotoxic (average IC50 = 5-10 µM) and possess high inhibitory activity (Ki = 1.25 nM) towards CAIX. Our results suggest that betulinyl sulfamates seem to be attractive substances, due to their possibility of targeted drug delivery they deserve to be proceeded for further pre-clinical (kinetic studies) and in vivo investigations.

A recessive mutation in beta-IV-spectrin (SPTBN4) associates with congenital myopathy, neuropathy, and central deafness.

Congenital myopathies are a heterogeneous group of muscle disorders that are often genetically determined. Here, we investigated a boy with congenital myopathy, deafness, and neuropathy from a consanguineous Kurdish family by autozygosity mapping and whole exome sequencing. We found a homozygous nonsense mutation in SPTBN4 [c.1597C>T, NM_020971.2; p.(Q533*), NP_066022.2; ClinVar SUB2292235] encoding ßIV-spectrin, a non-erythrocytic member of the ß-spectrin family. Western blot confirmed the absence of the full-length 288 kDa isoform in muscle and of a specific 72 kDa isoform in fibroblasts. Clinical symptoms of the patient largely corresponded to those described for the quivering mouse, a loss-of-function animal model. Since the human phenotype of ßIV-spectrin deficiency included a myopathy with incomplete congenital fiber-type disproportion, we investigated muscle of the quivering (qv4J) mouse and found complete absence of type 1 fibers (fiber-type 2 uniformity). Immunohistology confirmed expression of ßIV-spectrin in normal human and mouse muscle at the sarcolemma and its absence in patient and quivering (qv4J) mouse. SPTBN4 mRNA-expression levels in healthy skeletal muscle were found in the range of other regulatory proteins. More patients have to be described to confirm the triad of congenital myopathy, neuropathy and deafness as the defining symptom complex for ßIV-spectrin deficiency.

Characterization of a Dmd (EGFP) reporter mouse as a tool to investigate dystrophin expression.

BACKGROUND: Dystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining. RESULTS: We generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes. CONCLUSIONS: The novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.

Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures.

Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system.

Recessive DEAF1 mutation associates with autism, intellectual disability, basal ganglia dysfunction and epilepsy.

BACKGROUND: Various genetic defects cause autism associated with intellectual disability and epilepsy. Here, we set out to identify the genetic defect in a consanguineous Omani family with three affected children in whom mutations in known candidate genes had been excluded beforehand. METHODS: For mutation screening, we combined autozygosity mapping and whole exome sequencing. Segregation of potential disease variants with the phenotype was verified by Sanger sequencing. A splice-site mutation was confirmed and quantified by qPCR. RESULTS: We found an autosomal recessive splice acceptor mutation in DEAF1 (c.997+4A>C, p.G292Pfs*) in all affected individuals, which led to exon skipping, and reduced the normal full-length mRNA copy number in the patients to 5% of the wild-type level. Besides intellectual disability and autism, two of three affected siblings suffered from severe epilepsy. All patients exhibited dyskinesia of the limbs coinciding with symmetric T2 hyperintensities of the basal ganglia on cranial MRI. CONCLUSIONS: A recent report has shown dominant DEAF1 mutations to occur de novo in patients with intellectual disability. Here, we demonstrate that a DEAF1-associated disorder can also be inherited as an autosomal recessive trait with heterozygous individuals being entirely healthy. Our findings expand the clinical and genetic spectrum of DEAF1 mutations to comprise epilepsy and extrapyramidal symptoms.

Phosphate ions and glutaminyl cyclases catalyze the cyclization of glutaminyl residues by facilitating synchronized proton transfers.

Phosphate ions and glutaminyl cyclase (QC) both catalyze the formation of pyroglutamate (pE, pGlu) from N-terminal glutamine residues of peptides and proteins. Here, we studied the mechanism of glutamine cyclization using kinetic secondary deuterium and solvent isotope effects. The data suggest that proton transfer(s) are rate determining for the spontaneous reaction, and that phosphate and QC are accelerating the reaction by promoting synchronized proton transfers in a concerted mechanism. Thus, non-enzymatic and enzymatic catalysis of pyroglutamate formation exploit a similar mode of transition-state stabilization.

Cavin 1 function does not follow caveolar morphology.

The function of caveolae, small invaginations of the plasma membrane, remains a matter of debate. We discuss endocytosis and compartmentalization of metabolic and signaling pathways. Caveolin 3 (CAV3) and polymerase I and transcript release factor (PTRF) are important proteins that ensure shaping of caveolae in muscle cells. We investigated caveolae morphologically by electron microscopy in myotubes obtained from patients with CAV3 mutations and performed functional analyses in fibroblasts from a patient with a mutation in PTRF. Despite the complete clinical picture of a caveolinopathy, we found that caveolae in the CAV3-deficient myotubes were normal in shape and number. Furthermore, we found a difference in uptake of cholera toxin B between PTRF-deficient fibroblasts devoid of caveolae and normal fibroblasts. However, after caveolae were rescued by transfection of PTRF, cholera toxin B uptake did not normalize. We conclude that the presence of caveolae as an anatomic structure is not sufficient to ensure their proper function. Alternatively, the functional properties assigned to caveolae might be mediated by different mechanisms that have yet to be resolved.

Recessive truncating IGHMBP2 mutations presenting as axonal sensorimotor neuropathy.

OBJECTIVE: To identify the cause of sensorimotor neuropathy in a cohort of patients with genetically unsolved neuropathies (57 families with a total of 74 members) in whom hitherto known disease genes had been excluded. METHODS: We used autozygosity mapping or haplotype analysis to delineate potential disease loci in informative families. For mutation detection, we used either whole-exome sequencing or Sanger sequencing of positional candidates. Subsequently, a larger cohort was specifically screened for IGHMBP2 mutations. The pathogenicity of a splice-site mutation was verified in cultured patient skin fibroblasts on the messenger RNA level and by Western blot. RESULTS: We report on 5 patients with neuropathy from 3 families who carried truncating mutations in IGHMBP2. Contrary to the "classic" phenotype, they did not manifest with respiratory distress, but with progressive sensorimotor neuropathy. Only one patient required nocturnal mask ventilation, while 4 others maintained normal respiratory function by the age of 14, 18, 22, and 37 years. Three patients were still able to walk independently. All patients had a predominantly axonal sensorimotor neuropathy with subsequent muscle atrophy, but without obvious sensory symptoms. Two patients had signs of autonomic neuropathy. CONCLUSIONS: Mutations in IGHMBP2 should be considered in the molecular genetic workup of patients with hereditary sensorimotor neuropathies, even in the absence of respiratory symptoms.

Recessive REEP1 mutation is associated with congenital axonal neuropathy and diaphragmatic palsy.

OBJECTIVE: To identify the underlying genetic cause of a congenital neuropathy in a 5-year-old boy as part of a cohort of 32 patients from 23 families with genetically unresolved neuropathies. METHODS: We used autozygosity mapping coupled with next-generation sequencing to investigate a consanguineous family from Lebanon with 1 affected and 2 healthy children. Variants were investigated for segregation in the family by Sanger sequencing. A splice site mutation was further evaluated on the messenger RNA level by quantitative reverse transcription PCR. Subsequently, a larger cohort was specifically screened for receptor expression-enhancing protein 1 (REEP1) gene mutations. RESULTS: We detected a homozygous splice donor mutation in REEP1 (c.303+1-7GTAATAT>AC, p.F62Kfs23*; NM_022912) that cosegregated with the phenotype in the family, leading to complete skipping of exon 4 and a premature stop codon. The phenotype of the patient is similar to spinal muscular atrophy with respiratory distress type 1 (SMARD1) with additional distal arthrogryposis and involvement of the upper motor neuron manifested by pronounced hyperreflexia. CONCLUSION: To date, only dominant REEP1 mutations have been reported to be associated with a slowly progressive hereditary spastic paraplegia. The findings from our patient expand the phenotypical spectrum and the mode of inheritance of REEP1-associated disorders. Recessive mutations in REEP1 should be considered in the molecular genetic workup of patients with a neuromuscular disorder resembling SMARD1, especially if additional signs of upper motor neuron involvement and distal arthrogryposis are present.

A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues.

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aß) deposited in Alzheimer's disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aß N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day(-1), peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (kD2O/kH2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.