Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases implicated in antiviral defense systems, tumorigenesis, and protein quality control. TRIM proteins contribute to protein quality control by regulating the ubiquitin-proteasome system, endoplasmic reticulum-associated degradation, and macroautophagy/autophagy. However, the detailed mechanisms through which various TRIM proteins regulate downstream events have not yet been fully elucidated. Herein, we identified a novel function of TRIM22 in the regulation of autophagy. TRIM22 promotes autophagosome-lysosome fusion by mediating the association of GABARAP family proteins with PLEKHM1, thereby inducing the autophagic clearance of protein aggregates, independent of its E3 ubiquitin ligase activity. Furthermore, a TRIM22 variant associated with early-onset familial Alzheimer disease interferes with autophagosome-lysosome fusion and autophagic clearance. These findings suggest TRIM22 as a critical autophagic regulator that orchestrates autophagosome-lysosome fusion by scaffolding autophagy-related proteins, thus representing a potential therapeutic target in neurodegenerative diseases.<b>Abbreviations:</b> AD: Alzheimer disease; ADAOO: AD age of onset; AICD: APP intracellular domain; APP: amyloid beta precursor protein; BSA: bovine serum albumin; cDNAs: complementary DNAs; CQ: chloroquine; CTF: carboxyl-terminal fragment; EBSS: Earle's balanced salt solution; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; HA: hemagglutinin; HOPS: homotypic fusion and protein sorting; IFN: interferon; IL1A/IL-1α: interleukin 1 alpha; KO: knockout; MTORC1: mechanistic target of rapamycin kinase complex 1; NFKBIA/IκBα: NFKB inhibitor alpha; NFE2L2/NRF2: NFE2 like bZIP transcription factor; PBS: phosphate-buffered saline; PI3K: class I phosphoinositide 3-kinase; PLA: proximity ligation assay; PLEKHM1: pleckstrin homology and RUN domain containing M1; PSEN1: presenilin 1; SEM: standard errors of the means; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SNCA: synuclein alpha; SNP: single nucleotide polymorphism; TBS: tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TRIM: tripartite motif; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Splicing factor B subunit 4 (SF3B4), a component of the U2-pre-mRNA spliceosomal complex, contributes to tumorigenesis in several types of tumors. However, the oncogenic potential of SF3B4 in lung cancer has not yet been determined. The <i>in vivo</i> expression profiles of SF3B4 in non-small cell lung cancer (NSCLC) from publicly available data revealed a significant increase in SF3B4 expression in tumor tissues compared to that in normal tissues. The impact of SF3B4 deletion on the growth of NSCLC cells was determined using a siRNA strategy in A549 lung adenocarcinoma cells. SF3B4 silencing resulted in marked retardation of the A549 cell proliferation, accompanied by the accumulation of cells at the G0/G1 phase and increased expression of p27, p21, and p53. Double knockdown of SF3B4 and p53 resulted in the restoration of p21 expression and partial recovery of cell proliferation, indicating that the p53/p21 axis is involved, at least in part, in the SF3B4-mediated regulation of A549 cell proliferation. We also provided ubiquitination factor E4B (UBE4B) is essential for p53 accumulation after SF3B4 depletion based on followings. First, co-immunoprecipitation showed that SF3B4 interacts with UBE4B. Furthermore, UBE4B levels were decreased by SF3B4 depletion. UBE4B depletion, in turn, reproduced the outcome of SF3B4 depletion, including reduction of polyubiquitinated p53 levels, subsequent induction of p53/p21 and p27, and proliferation retardation. Collectively, our findings indicate the important role of SF3B4 in the regulation of A549 cell proliferation through the UBE4B/p53/p21 axis and p27, implicating the therapeutic strategies for NSCLC targeting SF3B4 and UBE4B.

Mutations in <i>WASHC5</i> (also known as <i>KIAA0196</i>) cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG8. WASHC5, commonly called strumpellin, is a core component of the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex that activates actin nucleation at endosomes. Although various other cellular roles for strumpellin have also been described, none account for how SPG8-associated mutations lead to HSP. Here, we identified protein interactors of the WASH complex by immunoprecipitation and mass spectrometry and assessed the functions of strumpellin in cultured cells using both overexpression and RNA interference along with cell-spreading assays to investigate cell adhesion. We uncovered a decrease in CAV1 protein abundance as well as endosomal fission defects resulting from pathogenic SPG8 mutations. CAV1, a key component of caveolae, interacted with strumpellin in cells, and strumpellin inhibited the lysosomal degradation of CAV1. SPG8-associated missense mutations in strumpellin did not rescue endosomal tubulation defects, reduction in CAV1 protein abundance, or integrin-mediated cell adhesion in strumpellin-deficient cells. Mechanistically, we demonstrated that the WASH complex maintained CAV1 and integrin protein amounts by inhibiting their lysosomal degradation through its endosomal actin nucleation activity. In addition, the interaction of strumpellin with CAV1 stimulated integrin recycling, thereby promoting cell adhesion. These findings provide a molecular link between <i>WASHC5</i> mutations and impairment of CAV1- and integrin-mediated cell adhesion, providing insights into the cellular pathogenesis of SPG8.

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases implicated in antiviral defense systems, tumorigenesis, and protein quality control. TRIM proteins contribute to protein quality control by regulating the ubiquitin-proteasome system, endoplasmic reticulum-associated degradation, and macroautophagy/autophagy. However, the detailed mechanisms through which various TRIM proteins regulate downstream events have not yet been fully elucidated. Herein, we identified a novel function of TRIM22 in the regulation of autophagy. TRIM22 promotes autophagosome-lysosome fusion by mediating the association of GABARAP family proteins with PLEKHM1, thereby inducing the autophagic clearance of protein aggregates, independent of its E3 ubiquitin ligase activity. Furthermore, a TRIM22 variant associated with early-onset familial Alzheimer disease interferes with autophagosome-lysosome fusion and autophagic clearance. These findings suggest TRIM22 as a critical autophagic regulator that orchestrates autophagosome-lysosome fusion by scaffolding autophagy-related proteins, thus representing a potential therapeutic target in neurodegenerative diseases.<b>Abbreviations:</b> AD: Alzheimer disease; ADAOO: AD age of onset; AICD: APP intracellular domain; APP: amyloid beta precursor protein; BSA: bovine serum albumin; cDNAs: complementary DNAs; CQ: chloroquine; CTF: carboxyl-terminal fragment; EBSS: Earle's balanced salt solution; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; HA: hemagglutinin; HOPS: homotypic fusion and protein sorting; IFN: interferon; IL1A/IL-1α: interleukin 1 alpha; KO: knockout; MTORC1: mechanistic target of rapamycin kinase complex 1; NFKBIA/IκBα: NFKB inhibitor alpha; NFE2L2/NRF2: NFE2 like bZIP transcription factor; PBS: phosphate-buffered saline; PI3K: class I phosphoinositide 3-kinase; PLA: proximity ligation assay; PLEKHM1: pleckstrin homology and RUN domain containing M1; PSEN1: presenilin 1; SEM: standard errors of the means; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SNCA: synuclein alpha; SNP: single nucleotide polymorphism; TBS: tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TRIM: tripartite motif; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Splicing factor B subunit 4 (SF3B4), a component of the U2-pre-mRNA spliceosomal complex, contributes to tumorigenesis in several types of tumors. However, the oncogenic potential of SF3B4 in lung cancer has not yet been determined. The <i>in vivo</i> expression profiles of SF3B4 in non-small cell lung cancer (NSCLC) from publicly available data revealed a significant increase in SF3B4 expression in tumor tissues compared to that in normal tissues. The impact of SF3B4 deletion on the growth of NSCLC cells was determined using a siRNA strategy in A549 lung adenocarcinoma cells. SF3B4 silencing resulted in marked retardation of the A549 cell proliferation, accompanied by the accumulation of cells at the G0/G1 phase and increased expression of p27, p21, and p53. Double knockdown of SF3B4 and p53 resulted in the restoration of p21 expression and partial recovery of cell proliferation, indicating that the p53/p21 axis is involved, at least in part, in the SF3B4-mediated regulation of A549 cell proliferation. We also provided ubiquitination factor E4B (UBE4B) is essential for p53 accumulation after SF3B4 depletion based on followings. First, co-immunoprecipitation showed that SF3B4 interacts with UBE4B. Furthermore, UBE4B levels were decreased by SF3B4 depletion. UBE4B depletion, in turn, reproduced the outcome of SF3B4 depletion, including reduction of polyubiquitinated p53 levels, subsequent induction of p53/p21 and p27, and proliferation retardation. Collectively, our findings indicate the important role of SF3B4 in the regulation of A549 cell proliferation through the UBE4B/p53/p21 axis and p27, implicating the therapeutic strategies for NSCLC targeting SF3B4 and UBE4B.

Mutations in <i>WASHC5</i> (also known as <i>KIAA0196</i>) cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG8. WASHC5, commonly called strumpellin, is a core component of the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex that activates actin nucleation at endosomes. Although various other cellular roles for strumpellin have also been described, none account for how SPG8-associated mutations lead to HSP. Here, we identified protein interactors of the WASH complex by immunoprecipitation and mass spectrometry and assessed the functions of strumpellin in cultured cells using both overexpression and RNA interference along with cell-spreading assays to investigate cell adhesion. We uncovered a decrease in CAV1 protein abundance as well as endosomal fission defects resulting from pathogenic SPG8 mutations. CAV1, a key component of caveolae, interacted with strumpellin in cells, and strumpellin inhibited the lysosomal degradation of CAV1. SPG8-associated missense mutations in strumpellin did not rescue endosomal tubulation defects, reduction in CAV1 protein abundance, or integrin-mediated cell adhesion in strumpellin-deficient cells. Mechanistically, we demonstrated that the WASH complex maintained CAV1 and integrin protein amounts by inhibiting their lysosomal degradation through its endosomal actin nucleation activity. In addition, the interaction of strumpellin with CAV1 stimulated integrin recycling, thereby promoting cell adhesion. These findings provide a molecular link between <i>WASHC5</i> mutations and impairment of CAV1- and integrin-mediated cell adhesion, providing insights into the cellular pathogenesis of SPG8.

“Green” emission: A novel two-photon fluorescent probe that can detect intracellular free Mg2+ ions at a depth of a few hundred micrometers in live tissue was developed. The emission from the Mg2+–probe complex (green) shows little interference from association of the probe with other intracellular metal ions or with the cell membrane (blue).

KRIBB11 induces apoptotic cell death in A172 cells through two axes: HSF1-dependent p53/p21 accumulation and Cdh1/SKP2-dependent reduction of p27.

Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.