Simpson-Louredo L et al. (2019) Antonie Van Leeuwenhoek "Detection and virulence potential of a phospholipase D-negative Corynebacterium ulcerans from a concurrent diphtheria and infectious mononucleosis case."

Burkovski A, Santana MM, Antunes CA, Silva CMF, Mattos-Guaraldi AL, Hirata R, Villas Boas MHS, Hacker E, Souza NF, Nagao PE, Simpson-Louredo L

[Antonie Van Leeuwenhoek, 2019]

Diphtheria byCorynebacterium ulceransis increasingly occurring inchildren, adolescents and adults.In addition todiphtheria toxin(DT), phospholipase D (PLD) is considered a virulence factor ofC. ulcerans. In the present study,a first case ofconcurrent diphtheria bya PLD-negative C. ulceransand infectious mononucleosis (IM) was verified. Clinical and microbiological profiles and binding properties to human Fibrinogen (Fbg), Fibronectin (Fn) and type I collagen (col I) biotinylated proteins and virulence to Caenorhabditis elegans were investigated for C. ulcerans strain 2590 (clinical isolate) and two control strains, including PLD-positive BR-AD22 wild type and PLD-negative ELHA-1 PLD mutant strains. MALDI-TOF assays and a multiplex PCR of genes coding for potentially toxigenic corynebacteria identified strain 2590 as non-DT producing. Interestingly, strain 2590 did not express PLD activity in the CAMP test although the presence ofthe pldgene was verified. PLD-negative 2590 and a PLD-positive 210932 strains showed similar affinity to Fbg, Fn and type I collagen. C. eleganswere able to escape fromC. ulcerans strains, independent of PLD and DT production.Higher mortality of nematodes was verified for PLD-negative strains. Additional studies concerning multifactorial virulence potential of C. ulcerans, including environmental conditions remain necessary.

Greene D et al. (2008) Biophys J "Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains."

Gernert KM, Benian GM, Sutton B, Garcia T, Greene D, Oberhauser AF

[Biophys J, 2008]

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the C. elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn beta-sandwich domains (30-150pN). Further, our AFM data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.

Diaz-Balzac CA et al. (2015) Cell Rep "The Adhesion Molecule KAL-1/anosmin-1 Regulates Neurite Branching through a SAX-7/L1CAM-EGL-15/FGFR Receptor Complex."

Lazaro-Pena MI, Bulow HE, Ramos-Ortiz GA, Diaz-Balzac CA

[Cell Rep, 2015]

Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system.

von Castelmur E et al. (2012) Proc Natl Acad Sci U S A "Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase."

Strumpfer J, Benian GM, Qadota H, Mayans O, Bogomolovas J, Franke B, Konarev PV, Schulten K, von Castelmur E, Labeit S, Barbieri S, Svergun DI

[Proc Natl Acad Sci U S A, 2012]

Titin-like kinases are an important class of cytoskeletal kinases that intervene in the response of muscle to mechanical stimulation, being central to myofibril homeostasis and development. These kinases exist in autoinhibited states and, allegedly, become activated during muscle activity by the elastic unfolding of a C-terminal regulatory segment (CRD). However, this mechano-activation model remains controversial. Here we explore the structural, catalytic, and tensile properties of the multidomain kinase region of Caenorhabditis elegans twitchin (Fn(31)-Nlinker-kinase-CRD-Ig(26)) using X-ray crystallography, small angle X-ray scattering, molecular dynamics simulations, and catalytic assays. This work uncovers the existence of an inhibitory segment that flanks the kinase N-terminally (N-linker) and that acts synergistically with the canonical CRD tail to silence catalysis. The N-linker region has high mechanical lability and acts as the primary stretch-sensor in twitchin kinase, while the CRD is poorly responsive to pulling forces. This poor response suggests that the CRD is not a generic mechanosensor in this kinase family. Instead, the CRD is shown here to be permissive to catalysis and might protect the kinase active site against mechanical damage. Thus, we put forward a regulatory model where kinase inhibition results from the combined action of both N- and C-terminal tails, but only the N-terminal extension undergoes mechanical removal, thereby affording partial activation. Further, we compare invertebrate and vertebrate titin-like kinases and identify variations in the regulatory segments that suggest a mechanical speciation of these kinase classes.