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Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons.

ABSTRACT: An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin C-terminal tail to interact with effector proteins and are thereby thought to change microtubule dynamics, stability, and organization. The peptidase(s) that produces ΔC2-α-tubulin in a physiological context remains unclear. Here, we take advantage of the observation that ΔC2-α-tubulin accumulates to high levels in cells lacking tubulin tyrosine ligase (TTL) to screen for cytosolic carboxypeptidases (CCPs) that generate ΔC2-α-tubulin. We identify CCP1 as the sole peptidase that produces ΔC2-α-tubulin in TTLΔ HeLa cells. Interestingly, we find that the levels of ΔC2-α-tubulin are only modestly reduced in photoreceptors of ccp1^-/- mice, indicating that other peptidases act synergistically with CCP1 to produce ΔC2-α-tubulin in post-mitotic cells. Moreover, the production of ΔC2-α-tubulin appears to be under tight spatial control in the photoreceptor cilium: ΔC2-α-tubulin persists in the connecting cilium of ccp1^-/- but is depleted in the distal portion of the photoreceptor. This work establishes the groundwork to pinpoint the function of ΔC2-α-tubulin in proliferating and post-mitotic mammalian cells.

SUBMITTER: Hotta T

PROVIDER: S-EPMC9952995 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons.

Hotta Takashi T Plemmons Alexandra A Gebbie Margo M Ziehm Trevor A TA Blasius Teresa Lynne TL Johnson Craig C Verhey Kristen J KJ Pearring Jillian N JN Ohi Ryoma R

Biomolecules 20230212 2

An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin C-terminal tail to interact with effector proteins and are thereby thought to change microtubule dynamics, stability, and organization. The peptidase(s) that produces ΔC2-α-tubulin ...[more]

PMID: 36830726

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Project description:The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the biology of neurons including cell death, survival, growth, differentiation, and plasticity. Regulation of the local availability of BDNF/TrkB at distinct subcellular domains such as soma, dendrites, axons, growth cones, nerve terminals, and spines appears to contribute to their specific functions. In view of the variance in size and shape of neurons and their compartments, previous quantitative studies of the BDNF/TrkB protein and mRNA lacked a robust normalization procedure. To overcome this problem, we have established methods that use immunofluorescence detection of α-tubulin as a normalization factor for the quantitative analysis of protein and mRNA in primary rat cortical and striatal neurons in culture. The efficacy of this approach is demonstrated by studying the dynamic distribution of proteins and mRNA at different growth stages or conditions. Treatment of cultured neurons with KCl resulted in increased levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple transcripts) and a slight reduction in BDNF protein levels in the dendrites from the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB proteins in the soma indicative of protein transport. Finally, analysis of the rat cortical and striatal neurons demonstrated comparable or even higher levels of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in contrast to previous observations made in vivo, striatal neurons are capable of synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical approach presented here provides a detailed understanding of BDNF/TrkB levels in response to a variety of neuronal activities. Our methods could be used broadly, including applications in cell and tissue cytometry, to yield accurate quantitative data of gene expression in cellular and subcellular contexts. © 2012 International Society for Advancement of Cytometry.

Dataset Information

Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons.

Publications

Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons.

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