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Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein–protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.

It has been 40 years since Francis Crick [ 1 ] noted the problem molecular turnover poses for maintaining memories and offered a general solution. The solution requires that the critical molecules must be replaced without altering the overall structure of the complex. It is timely then that Todd Sacktor’s group [ 2 ] has identified critical intermolecular interactions that satisfy Crick’s requirement. Sacktor’s early work identified the continuously active kinase, protein kinase Mzeta (PKMzeta) as a critical molecule for maintaining localized postsynaptic AMPA receptors that support long-term potentiation (LTP) and memory. More recent work revealed that PKMzeta forms heterodimers with the scaffolding protein KIBRA (KIbra BRAin) and preventing dimerization erased both LTP and memory. Even so, dimers degrade too fast to support long-lasting memories. Based on biophysical modeling, Sacktor’s group with Harel Shouval reasoned that if KIBRA-PKMzeta heterodimers interact to form oligomers (such as hexamers), they can survive molecular turnover because as a dimer degrades it can be replaced by another. AlphaFold 3 predicted a site where the small molecule inhibitor, zeta-stat, would bind and disrupt oligomer formation. If so, then infusing zeta-stat into the hippocampus should erase long-term memory. This predicted outcome was observed. Thus, Crick’s solution has been achieved. Oligomers formed from KIBRA-PKMzeta dimers allow degraded individual molecules to be replaced one at a time while maintaining their overall structure. This permits a continuous presence of PKMzeta where it interacts with AMPA receptors (through GluA2 subunits) and other molecules to ensure long-term memories endure.

Despite widespread discussion of the impact of corporate social responsibility (CSR) activities on consumer perceptions, little research has examined how consumers cope with CSR-based crisis response messages as a bolstering strategy. To fill this gap, we propose a framework integrating situational crisis communication theory with the persuasion knowledge model, applying the model to an experiment with a 2 (topic knowledge - crisis type: accidental vs. intentional) × 2 (persuasion knowledge- CSR motives: intrinsic vs. extrinsic) × 2 (agent knowledge- CSR history: long vs. short) between-subjects factorial design. In Study 1, we found interaction effects between CSR motives and crisis type on word-of-mouth intention and purchase intention. In addition, inferences about CSR motives interacted with perceptions about CSR history on purchase intention. In Study 2, we replicated study 1 and found that crisis responsibility mediated the main effect of crisis type on behavioral intentions, but neither the main effect of CSR motives and CSR history nor the interactions effects among those variables were mediated by crisis responsibility. Our results indicate that consumer inferences from a company's CSR-based crisis communications play a significant role in increasing consumer behavioral intentions in two situations: when a crisis is accidental and when a CSR history is short. Ethical and theoretical implications are discussed.

IntroductionFalse contextual fear memory has been attributed to a time-dependent loss of precision in contextual memory representations. In this study, we investigated the role of protein kinase M zeta (PKMζ), a key molecule in the maintenance of hippocampus-dependent long-term memory, in false contextual fear memory within the dorsal (dHPC) and ventral hippocampus (vHPC).MethodsTwo weeks prior to behavioral testing, male C57BL/6J mice (7–10 weeks old) received bilateral injections of adeno-associated virus (AAV PHP.eB) into the dHPC or vHPC to induce PKMζ knockdown (PKMζ KD), overexpression of wild-type PKMζ (PKMζ WT), or kinase-inactive PKMζ (PKMζ K281R). False contextual fear memory was assessed by measuring freezing behavior in Context B at 3 and 24 h following exposure to Context A with or without unconditioned stimulus presentation [US(+) and US(−), respectively]. Spatial recognition memory was evaluated using the two-trial novel arm recognition test in the Y-maze.ResultsAs shown in our previous work, mice exhibited freezing in Context B after receiving a US in Context A, whereas mice that did not receive such a stimulus showed minimal freezing. These data confirmed that freezing in Context B reflects false contextual fear memory. False fear responses were evident at 3 h and were further increased at 24 h. Freezing at 24 h was markedly enhanced in dHPC PKMζ knockdown mice compared with that in AAV control-injected mice. PKMζ WT overexpression prevented the increase in freezing at 24 h, whereas PKMζ K281R overexpression mimicked the effects of PKMζ KD. Furthermore, PKMζ knockdown in the dHPC impaired spatial recognition memory, indicating that hippocampus-dependent spatial processing was disrupted. In contrast, PKMζ manipulation in the vHPC did not affect false contextual fear memory but did impair spatial recognition memory.DiscussionThese findings are consistent with the possibility that functional loss of PKMζ in the dHPC affects contextual memory processes in a manner that may contribute to the time-dependent increase in false contextual fear and impaired spatial recognition memory.

LTP is the most intensively studied cellular model of the memory and generally divided at least two distinct phases as early and late. E-LTP requires activation of CaMKII that initiates biochemical events and trafficking of proteins, which eventually potentiate synaptic transmission, and is independent of de novo protein synthesis. In contrast, L-LTP requires gene expression and local protein synthesis regulated via TrkB receptor- and functional prions CPEB2-3-mediated translation. Maintenance of LTP for longer periods depends on constitutively active PKMζ. Throughout this review, current knowledge about early and late phases of LTP will be reviewed.

Consolidation and reconsolidation are independent memory processes. Consolidation stabilizes new memories, whereas reconsolidation restabilizes memories destabilized when reactivated during recall. However, the biological role of the destabilization/reconsolidation cycle is still unknown. It has been hypothesized that reconsolidation links new information with reactivated memories, but some reports suggest that new and old memories are associated through consolidation mechanisms instead. Object-recognition memory (ORM) serves to judge the familiarity of items and is essential for remembering previous events. We took advantage of the fact that ORM consolidation, destabilization, and reconsolidation can be pharmacologically dissociated to demonstrate that, depending on the activation state of hippocampal dopamine D1/D5 receptors, the memory of a novel object presented during recall of the memory of a familiar one can be formed via reconsolidation or consolidation, but only reconsolidation can link them. We also found that recognition memories formed through reconsolidation can be destabilized even if indirectly reactivated. Our results indicate that dopamine couples novelty detection with memory destabilization to determine whether a new recognition trace is associated with an active network and suggest that declarative reminders should be used with caution during reconsolidation-based psychotherapeutic interventions.

PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice. How are long-term memories stored in the brain? The formation of memories is believed to depend on the strengthening of connections between neurons. During learning, neurons produce an enzyme called PKMzeta (or PKMζ), which is thought to be responsible for maintaining the newly strengthened connections. Inhibitors of PKMzeta, such as a drug called ZIP, disrupt long-term memories. This suggests that the brain may be like a computer hard disc in that its stored information — its memories — could be erased. However, recent experiments on genetically engineered mice have thrown the role of PKMzeta into question. Knockout mice that lack the gene for PKMzeta can still strengthen connections between neurons and can still learn and remember. Moreover, ZIP still works to reverse the strengthening and to erase long-term memories. This indicates that ZIP can act on something other than the PKMzeta enzyme. These results have led many neuroscientists to doubt that PKMzeta has anything to do with memory. Yet there are two possible explanations for the normal memory in PKMzeta knockout mice. First, PKMzeta is not required for memory, so getting rid of it has no effect. Second, PKMzeta is essential for long-term memory in normal mice. However, knockout mice recruit a back-up mechanism for long-term memory storage, which is also sensitive to the effects of ZIP. To test these possibilities, Tsokas et al. used a modified piece of DNA that prevents neurons with the gene for PKMzeta from producing the enzyme. The DNA blocked memory formation in normal mice, consistent with a role for PKMzeta in memory. However, it had no effect in knockout mice — the DNA had nothing to work on. This suggests that another molecule does indeed act as a back-up for PKMzeta in these animals. Further experiments revealed that an enzyme closely related to PKMzeta, called PKCiota/lambda (PKCι/λ), substitutes for PKMzeta during memory storage in the knockout mice. These findings restore PKMzeta to its early promise. They show that PKMzeta is crucial for long-term memory in normal mice, but that something as important as memory storage has a back-up mechanism should PKMzeta fail. Future work may reveal when and how this back-up becomes engaged.

Objectives PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate‐limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. Materials and methods In this study, we used CRISPR‐based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA‐seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. Results The splicing sites of PKM, a 5’ donor site of GT and a 3’ acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax‐NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5’ donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non‐canonical PKM isoforms and produced some novel splicing isoforms. Conclusions This work proved that CRISPR‐based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases. Because of the lack of appropriate techniques, the specific functions of the two PKM splicing isoforms have not been clarified endogenously yet. In this study, we used CRISPR‐based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. It's proved that CRISPR‐based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo

Understanding of the microRNAs (miRNAs) regulatory system has become indispensable for physiological/oncological research. Tissue and organ specificities are key features of miRNAs that should be accounted for in cancer research. Further, cancer‐specific energy metabolism, referred to as the Warburg effect, has been positioned as a key cancer feature. Enhancement of the glycolysis pathway in cancer cells is what primarily characterizes the Warburg effect. Pyruvate kinase M1/2 (PKM1/2) are key molecules of the complex glycolytic system; their distribution is organ‐specific. In fact, PKM2 overexpression has been detected in various cancer cells. PKM isoforms are generated by alternative splicing by heterogeneous nuclear ribonucleoproteins. In addition, polypyrimidine tract‐binding protein 1 (PTBP1) is essential for the production of PKM2 in cancer cells. Recently, several studies focusing on non‐coding RNA elucidated PTBP1 or PKM2 regulatory mechanisms, including control by miRNAs, and their association with cancer. In this review, we discuss the strong relationship between the organ‐specific distribution of miRNAs and the expression of PKM in the context of PTBP1 gene regulation. Moreover, we focus on the impact of PTBP1‐targeting miRNA dysregulation on the Warburg effect. In this review, we discuss the miRNA‐mediated regulation of PTBP1 and PKM isoforms. In particular, we elaborate on the following points. First, under physiological conditions, the expression of PTBP1 and PKM isoforms is regulated by miRNAs that are unevenly distributed throughout the organs. Second, during carcinogenesis, dysregulation of PTBP1‐targeting miRNAs affects cancer‐specific energy metabolism in various types of cancer cells through PKM2 upregulation.

Background Aerobic glycolysis is a tumor cell phenotype and a hallmark in cancer research. The alternative splicing of the pyruvate kinase M ( PKM ) gene regulates the expressions of PKM1/2 isoforms and the aerobic glycolysis of tumors. Polypyrimidine tract binding protein (PTBP1) is critical in this process; however, its impact and underlying mechanisms in colorectal cancer (CRC) remain unclear. This study aimed to investigate the role of PTBP1 crotonylation in CRC progression. Methods The crotonylation levels of PTBP1 in human CRC tissues and cell lines were analyzed using crotonylation proteomics and immunoprecipitation. The main crotonylation sites were identified by immunoprecipitation and immunofluorescent staining. The glycolytic capacities of CRC cells were evaluated by measuring the glucose uptake, lactate production, extracellular acidification rate, and glycolytic proton efflux rate. The role and mechanism of PTBP1 crotonylation in PKM alternative splicing were determined by Western blot, quantitative real-time PCR (RT-qPCR), RNA immunoprecipitation, and immunoprecipitation. The effects of PTBP1 crotonylation on the behaviors of CRC cells and CRC progression were assessed using CCK-8, colony formation, cell invasion, wound healing assays, xenograft model construction, and immunohistochemistry. Results The crotonylation level of PTBP1 was elevated in human CRC tissues compared to peritumor tissues. In CRC tissues and cells, PTBP1 was mainly crotonylated at K266 (PTBP1 K266-Cr), and lysine acetyltransferase 2B (KAT2B) acted as the crotonyltranferase. PTBP1 K266-Cr promoted glycolysis and lactic acid production, increasing the PKM2/PKM1 ratio in CRC tissues and cells. Mechanistically, PTBP1 K266-Cr enhanced the interaction of PTBP1 with heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNPA1/2), thus affecting the PKM alternative splicing. PTBP1 K266-Cr facilitated CRC cell proliferation, migration, and metastasis in vitro and in vivo. Pathologically, a high level of PTBP1 K266-Cr was associated with poor prognosis in CRC patients. Conclusions Crotonylation of PTBP1 coordinates tumor cell glycolysis and promotes CRC progression by regulating PKM alternative splicing and increasing PKM2 expression.