Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibularinduced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.

Ndr2/Stk38l encodes a protein kinase associated with the Hippo tumor suppressor pathway and is mutated in a naturally-occurring canine early retinal degeneration (erd). To elucidate the retinal functions of Ndr2 and its paralog Ndr1/Stk38, we generated Ndr1 and Ndr2 single knockout mice. Although retinal lamination appeared normal in these mice, Ndr deletion caused a subset of Pax6-positive amacrine cells to proliferate in differentiated retinas, while concurrently decreasing the number of GABAergic, HuD and Pax6-positive amacrine cells. Retinal transcriptome analyses revealed that Ndr2 deletion increased expression of neuronal stress genes and decreased expression of synaptic organization genes. Consistent with the latter, Ndr deletion dramatically reduced levels of Aak1, an Ndr substrate that regulates vesicle trafficking. Our findings indicate that Ndr kinases are important regulators of amacrine and photoreceptor cells and suggest that Ndr kinases inhibit the proliferation of a subset of terminally differentiated cells and modulate interneuron synapse function via Aak1.

Recombinant human mast cell chymase (rhChymase) was expressed in secreted form as an active enzyme in the SuperMan5 strain of GlycoSwitch® Pichia pastoris, which is engineered to produce proteins with (Man)5(GlcNAc)2 Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5 mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDSPAGE as a single band of 30 kDa and treatment with peptide N-glycosidase F decreased this to 25 kDa, consistent with the established properties of native human chymase (hChymase). Polyclonal antibodies against hChymase detected rhChymase by Western blot. Active site titration with Eglin C, a potent chymase inhibitor, quantified the concentration of purified active enzyme. Kinetic analyses with succinyl-Ala-Ala-Pro-Phe (suc-AAPF) p-nitroanilide and thiobenzyl ester synthetic substrates showed that heparin significantly reduced Km, whereas heparin effects on kcat were minor. Pure rhChymase with Asn-linked glycans closely resembles hChymase. This bioengineering approach avoided hyperglycosylation and provides a source of active rhChymase for other studies as well as a foundation for production of recombinant enzyme with human glycosylation patterns.

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK~X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (k cat /K M 5 6.83 3 10 6 M 21 sec 21) and DDDDR (k cat /K M 5 1.89 3 10 7 M 21 sec 21) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge-charge interactions in the extended substrate binding pocket.

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase “super substrates” that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.

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Cathespin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG has dual specificity for chymotrypsin‐like and tryspin‐like substrates. CatG is a poorly understood enzyme and is currently only commercially available as mature enzyme purified from human sources. The yeast Pichia pastoris is being used to express CatG to study its dual specificity and its C‐terminal processing. The full length (C‐terminus present) human CatG amino acid sequence was modified to remove one glycosylation site and eight dibasic sites to avoid potential cleavage by yeast kexin protease. The construct was engineered to have an N‐terminal 6‐His‐cytochrome B5 (CytB5) heme binding fusion domain linked to the modified human CatG by an enterokinase cleavage site for activation. The amino acid sequence was used to generate a codon‐optimized gene that was placed in the pPICzα secretion vector. After transforming Pichia pastoris strain X‐33, 48 Zeocin‐resistant clones were screened for relative levels of CatG activity. Recombinant CatG has been partially purified from fermentation media by nickel affinity chromatography and its activity has been confirmed by assays using synthetic substrates. Supported by a Student Faculty Collaborative Grant from the ETSU Honors College and ETSU Office of Research and Sponsored Programs and by NHLB grant R15HL091770.

The catalytic light chain of the serine protease human enteropeptidase (enterokinase, hEKLC) was expressed as an active recombinant protein in the yeast Pichia pastoris. EKLC shows high degrees of specificity and selectivity for the amino acid sequences DDDDK~X and DDDDR~X, making it useful for separating fusion domains of recombinant proteins. The amino acid sequence of hEKLC was altered to remove N‐linked glycosylation sites by changing Asn to Gln; similarly, an Arg in a potential kexin protease cleavage site was changed to Gln to preclude proteolysis. The cDNA encoding hEKLC was codon‐optimized for expression in P. pastoris and commercially synthesized. Fusion to α‐mating factor targeted the protein for secretion. Active recombinant hEKLC (rhEKLC) was purified from fermentation medium by liquid chromatography using the enzyme's affinity for soybean trypsin inhibitor (STI). Western blot results confirm the identity of rhEKLC using an antibody to bovine enteropeptidase. Enzymatic activity was characterized via cleavage of the synthetic substrates z‐Lys‐SBzl, GDDDDK‐p‐nitroanilide, and GDDDDR‐p‐nitroanilide. Since the enzyme is secreted in active form, its expression is useful for testing P. pastoris expression media and it has proven useful for activating other proteases expressed as fusion proteins linked via DDDDK sequences. NIH grant R15HL091770.