PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites. IMPORTANCE: Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

Post-translational regulation of the mTORC1 pathway: A switch that regulates metabolism-related gene expression.

The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that plays a crucial role in coordinating cell growth in response to various signals, including amino acids, growth factors, oxygen, and ATP. Activation of mTORC1 promotes cell growth and anabolism, while its suppression leads to catabolism and inhibition of cell growth, enabling cells to withstand nutrient scarcity and stress. Dysregulation of mTORC1 activity is associated with numerous diseases, such as cancer, metabolic disorders, and neurodegenerative conditions. This review focuses on how post-translational modifications, particularly phosphorylation and ubiquitination, modulate mTORC1 signaling pathway and their consequential implications for pathogenesis. Understanding the impact of phosphorylation and ubiquitination on the mTORC1 signaling pathway provides valuable insights into the regulation of cellular growth and potential therapeutic targets for related diseases.

Regulation of condensin II by self-suppression and release mechanisms.

In vertebrates, two distinct condensin complexes, condensin I and condensin II, cooperate to drive mitotic chromosome assembly. It remains largely unknown how the two complexes differentially contribute to this process at a mechanistic level. We have previously dissected the role of individual subunits of condensin II by introducing recombinant complexes into Xenopus egg extracts. Here we extend these efforts by introducing a modified functional assay using extracts depleted of topoisomerase IIα (topo IIα), which allows us to further elucidate the functional similarities and differences between condensin I and condensin II. The intrinsically disordered C-terminal region of the CAP-D3 subunit (the D3 C-tail) is a major target of Cdk1 phosphorylation, and phosphorylation-deficient mutations in this region impair condensin II functions. We also identify a unique helical structure in CAP-D3 (the D3 HEAT docker) that is predicted to directly interact with CAP-G2. Deletion of the D3 HEAT docker, along with the D3 C-tail, enhances the ability of condensin II to assemble mitotic chromosomes. Taken together, we propose a self-suppression mechanism unique to condensin II that is released by mitotic phosphorylation. Evolutionary implications of our findings are also discussed.

Bridging condensins mediate compaction of mitotic chromosomes.

Eukaryotic chromosomes compact during mitosis into elongated cylinders-and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamic simulations involving two types of such proteins with different activities. One, which we refer to as looping condensins, anchors long-lived chromatin loops to create bottlebrush structures. The second, referred to as bridging condensins, forms multivalent bridges between distant parts of these loops. We show that binding of bridging condensins leads to the formation of shorter and stiffer mitotic-like cylinders without requiring any additional energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes seen experimentally and points to a mechanistic basis for the structure of common fragile sites in mitotic chromosomes.

De novo missense variants in RRAGC lead to a fatal mTORopathy of early childhood.

PURPOSE: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell growth in response to nutritional status. Central to the mTORC1 function is the Rag-GTPase heterodimer. One component of the Rag heterodimer is RagC (Ras-related GTP-binding protein C), which is encoded by the RRAGC gene. METHODS: Genetic testing via trio exome sequencing was applied to identify the underlying disease cause in 3 infants with dilated cardiomyopathy, hepatopathy, and brain abnormalities, including pachygyria, polymicrogyria, and septo-optic dysplasia. Studies in patient-derived skin fibroblasts and in a HEK293 cell model were performed to investigate the cellular consequences. RESULTS: We identified 3 de novo missense variants in RRAGC (NM_022157.4: c.269C>A, p.(Thr90Asn), c.353C>T, p.(Pro118Leu), and c.343T>C, p.(Trp115Arg)), which were previously reported as occurring somatically in follicular lymphoma. Studies of patient-derived fibroblasts carrying the p.(Thr90Asn) variant revealed increased cell size, as well as dysregulation of mTOR-related p70S6K (ribosomal protein S6 kinase 1) and transcription factor EB signaling. Moreover, subcellular localization of mTOR was decoupled from metabolic state. We confirmed the key findings for all RRAGC variants described in this study in a HEK293 cell model. CONCLUSION: The above results are in line with a constitutive overactivation of the mTORC1 pathway. Our study establishes de novo missense variants in RRAGC as cause of an early-onset mTORopathy with unfavorable prognosis.

Knockdown of regulatory associated protein of TOR (raptor) in hypothalamus-stimulated folliculogenesis and induced ovarian cysts.

CONTEXT: Mammalian target of rapamycin complex 1 (mTORC1) is an essential sensor that regulates fundamental biological processes like cell growth, proliferation and energy metabolism. The treatment of disease by sirolimus, a mTORC1 inhibitor, causes adverse effects, such as female fertility disorders. AIMS: The objective of the study was to decipher the reproductive consequences of a downregulation of mTORC1 in the hypothalamus. METHODS: The reduced expression of mTORC1 was induced after intracerebroventricular injection of lentivirus expressing a short hairpin RNA (shRNA) against regulatory associated protein of TOR (raptor) in adult female mice (ShRaptor mice). KEY RESULTS: The ShRaptor mice were fertile and exhibited a 15% increase in the litter size compared with control mice. The histological analysis showed an increase in antral, preovulatory follicles and ovarian cysts. In the hypothalamus, the GnRH mRNA and FSH levels in ShRaptor mice were significantly elevated. CONCLUSIONS: These results support the hypothesis that mTORC1 in the central nervous system participates in the regulation of female fertility and ovarian function by influencing the GnRH neuronal activity. IMPLICATIONS: These results suggest that a lower mTORC1 activity directly the central nervous system leads to a deregulation in the oestrous cycle and an induction of ovarian cyst development.

Cystine Induced-mTORC2 Activation through Promoting Sin1 Phosphorylation to Suppress Cancer Cell Ferroptosis.

SCOPE: Mechanistic target of rapamycin (mTOR) serves as a central signaling node in the coordination of cell growth and metabolism, and it functions via two distinct complexes, namely, mTOR complex 1 (mTORC1) and mTORC2. mTORC1 plays a crucial role in sensing amino acids, whereas mTORC2 involves in sensing growth factors. However, it remains largely unclear whether mTORC2 can sense amino acids and the mechanism by which amino acids regulate mTORC2 has not been studied. METHODS AND RESULTS: After treating cells with indicated concentration of amino acids for different time, it is found that the mTORC2 activation is significantly increased in response to amino acids stimulation, especially cystine. Particularly, knockdown solute carrier family 7 member 11 (SLC7A11) by siRNA shows that SLC7A11-mediated cystine uptake is responsible for activating mTORC2. Mechanistically, the study finds that p38 is activated in response to cystine stimulation, and co-immunoprecipitation (Co-IP) experiments suggest that p38 regulates the assembly of components within mTORC2 by mediating the phosphorylation of the mTORC2 subunit mitogen-activated protein kinase-interacting protein 1 (Sin1) in a cystine-dependent manner. Finally, combined with inducers and inhibitors of ferroptosis and cell viability assay, the study observes that cystine-mediated regulation of the p38-Sin1-mTOR-AKT pathway induces resistance to ferroptosis. CONCLUSION: These results indicate that cystine-induced activation of the p38-Sin1-mTORC2-AKT pathway suppresses ferroptosis.

Topoisomerases I and II facilitate condensin DC translocation to organize and repress X chromosomes in C. elegans.

Condensins are evolutionarily conserved molecular motors that translocate along DNA and form loops. To address how DNA topology affects condensin translocation, we applied auxin-inducible degradation of topoisomerases I and II and analyzed the binding and function of an interphase condensin that mediates X chromosome dosage compensation in C. elegans. TOP-2 depletion reduced long-range spreading of condensin-DC (dosage compensation) from its recruitment sites and shortened 3D DNA contacts measured by Hi-C. TOP-1 depletion did not affect long-range spreading but resulted in condensin-DC accumulation within expressed gene bodies. Both TOP-1 and TOP-2 depletion resulted in X chromosome derepression, indicating that condensin-DC translocation at both scales is required for its function. Together, the distinct effects of TOP-1 and TOP-2 suggest two distinct modes of condensin-DC association with chromatin: long-range DNA loop extrusion that requires decatenation/unknotting of DNA and short-range translocation across genes that requires resolution of transcription-induced supercoiling.

FRA1:c-JUN:HDAC1 complex down-regulates filaggrin expression upon TNFα and IFNγ stimulation in keratinocytes.

Filaggrin (FLG), an essential structural protein for skin barrier function, is down-regulated under chronic inflammatory conditions, leading to disruption of the skin barrier. However, the detailed molecular mechanisms of how FLG changes in the context of chronic inflammation are poorly understood. Here, we identified the molecular mechanisms by which inflammatory cytokines inhibit FLG expression in the skin. We found that the AP1 response element within the -343/+25 of the FLG promoter was necessary for TNFα + IFNγ-induced down-regulation of FLG promoter activity. Using DNA affinity precipitation assay, we observed that AP1 subunit composition binding to the FLG promoter was altered from c-FOS:c-JUN (at the early time) to FRA1:c-JUN (at the late time) in response to TNFα + IFNγ stimulation. Knockdown of FRA1 or c-JUN abrogated TNFα + IFNγ-induced FLG suppression. Histone deacetylase (HDAC) 1 interacted with FRA1:c-JUN under TNFα + IFNγ stimulation. Knockdown of HDAC1 abrogated the inhibitory effect of TNFα + IFNγ on FLG expression. The altered expression of FLG, FRA1, c-JUN, and HDAC1 was confirmed in mouse models of 2,4-dinitrochlorobenzene-induced atopic dermatitis and imiquimod-induced psoriasis. Thus, the current study demonstrates that TNFα + IFNγ stimulation suppresses FLG expression by promoting the FRA1:c-JUN:HDAC1 complex. This study provides insight into future therapeutic strategies targeting the FRA1:c-JUN:HDAC1 complex to restore impaired FLG expression in chronic skin inflammation.

Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition.

Mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.

The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity.

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.

Structural basis for SHOC2 modulation of RAS signalling.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers1-3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5-8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11-13, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation.

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Apo(a) and ApoB Interact Noncovalently Within Hepatocytes: Implications for Regulation of Lp(a) Levels by Modulation of ApoB Secretion.

BACKGROUND: Elevated plasma Lp(a) (lipoprotein(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease and aortic valve stenosis. However, the cell biology of Lp(a) biosynthesis remains poorly understood, with the locations of the noncovalent and covalent steps of Lp(a) assembly unclear and the nature of the apoB-containing particle destined for Lp(a) unknown. We, therefore, asked if apo(a) and apoB interact noncovalently within hepatocytes and if this impacts Lp(a) biosynthesis. METHODS: Using human hepatocellular carcinoma cells expressing 17K (17 kringle) apo(a), or a 17KΔLBS7,8 variant with a reduced ability to bind noncovalently to apoB, we performed coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays to document intracellular apo(a):apoB interactions. We used a pulse-chase metabolic labeling approach to measure apo(a) and apoB secretion rates. RESULTS: Noncovalent complexes containing apo(a)/apoB are present in lysates from cells expressing 17K but not 17KΔLBS7,8, whereas covalent apo(a)/apoB complexes are absent from lysates. 17K and apoB colocalized intracellularly, overlapping with staining for markers of endoplasmic reticulum trans-Golgi, and early endosomes, and less so with lysosomes. The 17KΔLBS7,8 had lower colocalization with apoB. Proximity ligation assays directly documented intracellular 17K/apoB interactions, which were dramatically reduced for 17KΔLBS7,8. Treatment of cells with PCSK9 (proprotein convertase subtilisin/kexin type 9) enhanced, and lomitapide reduced, apo(a) secretion in a manner dependent on the noncovalent interaction between apo(a) and apoB. Apo(a) secretion was also reduced by siRNA-mediated knockdown of APOB. CONCLUSIONS: Our findings explain the coupling of apo(a) and Lp(a)-apoB production observed in human metabolic studies using stable isotopes as well as the ability of agents that inhibit apoB biosynthesis to lower Lp(a) levels.

A loop extrusion-independent mechanism contributes to condensin I-mediated chromosome shaping.

Condensin I is a five-subunit protein complex that is central to mitotic chromosome assembly in eukaryotic cells. Despite recent progress, its molecular mechanisms of action remain to be fully elucidated. By using Xenopus egg extracts as a functional assay, we find that condensin I complexes harboring mutations in its kleisin subunit CAP-H produce chromosomes with confined axes in the presence of topoisomerase IIα (topo IIα) and highly compact structures (termed "beans") with condensin-positive central cores in its absence. The bean phenotype depends on the SMC ATPase cycle and can be reversed by subsequent addition of topo IIα. The HEAT repeat subunit CAP-D2, but not CAP-G, is essential for the bean formation. Notably, loop extrusion activities of the mutant complexes cannot explain the chromosomal defects they exhibit in Xenopus egg extracts, implying that a loop extrusion-independent mechanism contributes to condensin I-mediated chromosome assembly and shaping. We provide evidence that condensin-condensin interactions underlie these processes.

Unique protein interaction networks define the chromatin remodelling module of the NuRD complex.

The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1 and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodelling module of the nucleosome remodelling and deacetylase (NuRD) complex. To date, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential for defining underlying mechanisms of gene regulation. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS), we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodelling activity into both ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner. We define a short aggregation-prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the nuclear receptor co-repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

Keeping intracellular DNA untangled: A new role for condensin?

The DNA-passage activity of topoisomerase II accidentally produces DNA knots and interlinks within and between chromatin fibers. Fortunately, these unwanted DNA entanglements are actively removed by some mechanism. Here we present an outline on DNA knot formation and discuss recent studies that have investigated how intracellular DNA knots are removed. First, although topoisomerase II is able to minimize DNA entanglements in vitro to below equilibrium values, it is unclear whether such capacity performs equally in vivo in chromatinized DNA. Second, DNA supercoiling could bias topoisomerase II to untangle the DNA. However, experimental evidence indicates that transcriptional supercoiling of intracellular DNA boosts knot formation. Last, cohesin and condensin could tighten DNA entanglements via DNA loop extrusion (LE) and force their dissolution by topoisomerase II. Recent observations indicate that condensin activity promotes the removal of DNA knots during interphase and mitosis. This activity might facilitate the spatial organization and dynamics of chromatin.

Epigenetic Silencing of Recombinant Adeno-associated Virus Genomes by NP220 and the HUSH Complex.

The single-stranded DNA genome of adeno-associated viruses (AAV) undergoes second-strand synthesis and transcription in the host cell nucleus. While wild-type AAV genomes are naturally silenced upon integration into the host genome, recombinant AAV (rAAV) genomes typically provide robust expression of transgenes persisting as extrachromosomal DNA or episomes. Episomal DNA associating with host histones is subject to epigenetic modifications, although the mechanisms underlying such are not well understood. Here, we provide evidence that the double-stranded DNA binding protein NP220, in association with the human silencing hub (HUSH) complex, mediates transcriptional silencing of single-stranded as well as self-complementary rAAV genomes. In cells lacking NP220 or other components of the HUSH complex, AAV genome transcript levels are increased and correlate with a marked reduction in repressive H3K9 histone methylation marks. We also provide evidence that the AAV capsid (serotype) can profoundly influence NP220-mediated silencing of packaged genomes, indicating potential role(s) for capsid-genome or capsid-host factor interactions in regulating epigenetic silencing of rAAV genomes. IMPORTANCE Recombinant AAV vectors can enable long-term gene expression in a wide variety of tissues. However, transgene silencing has been reported in some human gene therapy clinical trials. Here, we demonstrate the HUSH complex can suppress transcript formation from rAAV vector genomes by epigenetic modification of associated host histones. Further, the AAV capsid appears to play an important role in this pathway. We postulate that modulation of epigenetic pathways could help improve rAAV expression.

PKM2 compensates for proteasome dysfunction by mediating the formation of the CHIP-HSP70-BAG3 complex and the aggregation of ubiquitinated proteins.

Protein aggregation and degradation via autophagy (aggrephagy) are major strategies adopted by cells to remove misfolded polypeptides when there is proteasome dysfunction. The functional protein complex consisting of heat shock protein 70 (Hsp70), cochaperone ubiquitin ligase carboxyl-terminal of Hsp70/Hsp90 interacting protein (CHIP), and co-chaperone Bcl-2-associated athanogene 3 (BAG3) has been associated with the activation of protein aggregation. However, data on the mechanisms of action of the complex in the protein degradation remains scant. Here, we report that upon proteasome stress, the M2 isoform of pyruvate kinase (PKM2) promotes the aggregation of ubiquitinated proteins and its knockout or knockdown aggravates the sensitivity of cells to proteasome inhibitors. Besides, following proteasome inhibition, PKM2 promotes the interaction of BAG3 with CHIP and HSP70. Interestingly, re-expression of loss-of-function mutants in PKM2-knockout cells showed that the regulatory function of PKM2 in this progress does not depend on the activity of glycolytic enzymes or protein kinases. Taken together, these findings demonstrate that PKM2 mediates the formation of the CHIP-HSP70-BAG3 protein complex and promotes the aggregation of ubiquitinated misfolded proteins, thus compensating for proteasome stress in cells.