Biology subjects

Lim, J. P.

Publications and source records attributed to Lim, J. P..

4 recordsLinked to original sources

bioRxiv2026

A distal CTCF-binding site drives MYC expression plasticity in a negative feed-forward loop

Oncogene expression heterogeneity systemically diversifies cancer cell phenotypes, enabling selection and cancer evolution. Here we document that the level of MYC expression variation is coordinated by a negative feed-forward loop involving the non-coding CCAT1 eRNA and the MYC gating process. While CCAT1 eRNA indirectly antagonizes MYC gating by promoting MYC transcriptional initiation and elongation, the MYC protein promotes gated MYC expression in a feedback loop by inhibiting both MYC transcription and CCAT1 expression. This entire process is coordinated by a CTCF binding site positioned within CCAT1 at a distal, oncogenic super-enhancer, which functions as a master switch by coordinating both CCAT1 and gated MYC expression to diversify cells toward both low and high MYC levels, as determined by heterogeneity metrics. As hallmarks of this principle are frequent in breast cancer and colorectal tumors, the dynamics of this new principle may underlie transitions between therapy-resistant low-MYC, and proliferative high-MYC tumor cells. Highlights- Transcriptional rate regulates MYC gating frequency - CCAT1 expression indirectly inhibits MYC gating by promoting MYC transcription - High MYC protein levels promote gated expression by inhibiting both MYC transcription and CCAT1 expression - These features are controlled by a single CTCF binding in the distant super-enhancer to drive expression plasticity in colorectal cancer cells

cancer biology↗

bioRxiv2022

N-Terminomic Changes of Neurons During Excitotoxicity Reveals Proteolytic Events Associated with Synaptic Dysfunctions and Potential Targets for Neuroprotection

Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase II{beta} (CaMKII{beta}). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbation of synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKII, an isoform of CaMKII{beta} undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection. In BriefAmeen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection. HighlightsO_LIIdentification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity. C_LIO_LIThe calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons. C_LIO_LIThese pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death. C_LIO_LISome of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection. C_LI Graphical abstract: Pathological proteolytic events in neurons during excitotoxicity unveiled by N-terminomic analyses(A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKII and CaMKII{beta}, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotection O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/484119v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1c69873org.highwire.dtl.DTLVardef@142db3forg.highwire.dtl.DTLVardef@481521org.highwire.dtl.DTLVardef@6359e2_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryProteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.

neuroscience↗

bioRxiv2020

An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

Excitotoxicity, a neuronal death process in neurological disorders, is initiated by over-stimulation of neuronal ionotropic glutamate receptors. The over-stimulated receptors dysregulate proteases, protein kinases and phosphatases, which in turn modify target neuronal proteins to induce cell death. To decipher this cell death mechanism, we used quantitative proteomics, phosphoproteomics and N-terminomics to identify modified proteins in excitotoxic neurons. Data, available in ProteomeXchange (identifiers: PXD019527 and PXD019211), enabled us to identify over one thousand such proteins with calpains, cathepsins and over twenty protein kinases as their major modifiers. These protein modification events can potentially perturb signalling pathways governing cell survival, synaptogenesis, axonal guidance and mRNA processing. Importantly, blocking the modification of Src protein kinase, a signalling hub in excitotoxic neurons, protected against neuronal loss in vivo in a rat model of neurotoxicity. Besides offering new insights into excitotoxic neuronal death mechanism, our findings suggest potential neuroprotective therapeutic targets for treating neurological disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/151456v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1f0191forg.highwire.dtl.DTLVardef@17ba2org.highwire.dtl.DTLVardef@15c0848org.highwire.dtl.DTLVardef@123d28b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMulti-dimensional proteomic analysis identified proteins modified by proteolysis and altered phosphorylation in neurons undergoing excitotoxic cell death. C_LIO_LICalpains, cathepsins and over twenty protein kinases are major modifiers of these proteins. C_LIO_LIThese protein modification events are predicted to impact cell survival, axonal guidance, synaptogenesis and mRNA processing. C_LIO_LIBlocking modification of an identified protein Src, which acts as a major signalling hub in neurons, was protective against excitotoxic injury in vivo. C_LI In BriefUsing multidimensional proteomic approaches, Ameen, et al. mapped the changes of proteome, phosphoproteome and N-terminome of cultured primary neurons during excitotoxicity, a crucial neuronal death process in neurological disorders. These proteomic changes document new excitotoxicity-associated molecular events, and offer insights into how these events are organized to induce neuronal death. Potential therapeutic relevance of these molecular events is illustrated by the demonstration that in vivo blockade of one of these events could protect against excitotoxic neuronal loss.

biochemistry↗

bioRxiv2017

Loss of CaMKI function disrupts salt aversive learning in C. elegans

The ability to adapt behavior to environmental fluctuations is critical for survival of organisms ranging from invertebrates to mammals. Caenorhabditis elegans can learn to avoid sodium chloride when it is paired with starvation. This behavior is likely advantageous to avoid areas without food. While some genes have been implicated in this salt aversive learning behavior, critical genetic components, and the neural circuit in which they act, remain elusive. Here, we show that the sole worm ortholog of mammalian CaMKI/IV, CMK-1, is essential for salt aversive learning behavior in C. elegans. We find that CMK-1 acts in the primary salt-sensing ASE neurons to regulate this behavior. By characterizing the intracellular calcium dynamics in ASE neurons using microfluidics, we find that loss of cmk-1 leads to an altered pattern of sensory-evoked calcium responses that may underlie salt aversive learning. Our study implicates the conserved CaMKI/CMK-1 as an essential cell-autonomous regulator for behavioral plasticity to environmental salt in C. elegans.\n\nSignificance StatementLike other animals, the nematode Caenorhabditis elegans depends on salt for survival and navigates toward high concentrations of this essential mineral. Because salt generates osmotic stress at high concentrations, it also threatens the survival of small terrestrial animals like C. elegans. A growing body of evidence indicates that C. elegans balances these factors through a process called salt aversive learning. We show that this behavior depends on expression of a calcium/calmodulin-dependent kinase, CMK-1, in the ASE salt sensing neurons and that salt-induced calcium signals in the ASE neurons are less sensitive to salt conditioning in animals lacking CMK-1 function. Our study identifies CMK-1 and sensory neurons as key factors in this form of behavioral plasticity.

neuroscience↗