Terje Johansen

Dual-specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) modulates the cell cycle and cell fate during development, and is deregulated in cancer and metabolic syndrome. However, only a few DYRK1B substrates have been defined, so we undertook a phosphoproteomics screen in cells that exhibit inducible DYRK1B expression. Motif analysis revealed enrichment for proline-directed serine or threonine phosphorylation sites (pSer–Pro or pThr–Pro), consistent with the consensus motif of class I DYRKs. Gene Ontology (GO) analysis revealed enrichment of proteins involved in mRNA binding, mRNA processing and ribonucleoprotein complexes. Several processing body (PB) components, including DCP1A, PATL1 (PAT1B), EDC3 and 4E-T (also known as EIF4ENIF1), were identified as DYRK1B-inducible phosphoproteins. DYRK1B also co-immunoprecipitated with DCP1A, PAT1B, EDC3, EDC4, DDX6 and XRN1. Super-resolution microscopy demonstrated that DYRK1B co-localised with DCP1A, DCP1B and DDX6 in PBs. Expression of DYRK1B increased PB abundance, whereas inhibition, depletion or knockout of DYRK1B reduced phosphorylation of DCP1A and 4E-T and decreased PB number. Re-expression of wild-type but not kinase-dead DYRK1B restored PB numbers in knockout cells. These findings reveal novel DYRK1B targets and establish DYRK1B as a regulator of PB abundance.

The selective autophagy receptors SQSTM1/p62 and NBR1 are evolutionary related and involved in autophagy of different substrates including proteins, protein aggregates and organelles. The two proteins interact via their PB1 domains, and essential for their roles in autophagy is the formation of a specific type of condensates named p62 bodies. The scaffold of these structures is formed by the interaction of polymeric p62 with polyubiquitin, but NBR1 is recruited and essential for their formation. Previous studies have shown that p62 contains nuclear export signal (NES) and nuclear localization signal (NLS) motifs and shuttles between the cytoplasm and the nucleus. Its nuclear roles are not fully understood, but there is evidence that p62 is involved in protein quality control in the nucleus. No previous studies have tested if NBR1 is transported into the nucleus. We show here that NBR1 contains two NES motifs and one NLS motif, and like p62, the protein shuttles between the cytoplasm and the nucleus. NBR1 also accumulates in nuclear p62 bodies and the formation of nuclear p62 bodies depends on NBR1.

Acute inflammation is an essential response that our bodies use to combat infections¹. However, in the absence of infections, chronic inflammation can have a pivotal role in the onset and progression of chronic diseases, such as arthritis, cancer, autoimmune disorders, metabolic-dysfunction-associated steatohepatitis (MASH), and most ageing-associated pathologies^2,3. The underlying mechanisms that distinguish chronic inflammation from its acute counterpart remain unclear, posing challenges to the development of targeted therapies for these major diseases. Here we identify a mechanism that separates the two responses: during chronic but not acute inflammation, chromatin remodelling is influenced by nuclear autophagy, in which the WSTF protein of the ISWI chromatin-remodelling complex interacts with the ATG8 autophagy protein family in the nucleus. This interaction leads to WSTF nuclear export and subsequent degradation by autophagosomes and lysosomes in the cytoplasm. Loss of WSTF leads to chromatin opening over inflammatory genes, amplifying inflammation. Cell-penetrating peptides that block the WSTF–ATG8 interaction do not affect acute inflammation but suppress chronic inflammation in senescence as well as in MASH and osteoarthritis in mouse models and patient samples. The ability to specifically target chronic inflammation without blunting acute inflammation offers an approach for treating common chronic inflammatory diseases.

Ammonia is a natural waste product of cellular metabolism which, through its lysosomotropic ability, can have detrimental effects on various cellular functions. Increased levels of ammonia were recently detected in the interstitial fluid of various tumours, substantiating that high ammonia concentrations are a pathophysiological condition in the tumour microenvironment, alongside hypoxia and acidosis. Since little is known about how cancer cells respond to elevated levels of ammonia in the tumour microenvironment, we investigated how a panel of cancer cell lines derived from solid tumours behaved when exposed to increasing concentrations of ammonia. We found that ammonia represses cell growth, induces genome instability, and inhibits lysosome-mediated proteolysis in a dose-dependent manner. Unexpectedly, we also found that small fluctuations in the pH of the extracellular environment, had a significant impact on the cytotoxic effects of ammonia. In summary, our data show that the balance of pH and ammonia within the interstitial fluids of cancerous tumours significantly impacts the behaviour and fate of cells residing in the tumour microenvironment.

Tumor protein p53 inducible nuclear protein 2 (TP53INP2; also known as DOR) is a multifunctional protein involved in transcriptional coactivation, ribosomal RNA synthesis and autophagy, regulated by subcellular localization. Using CRISPR/Cas9-generated TP53INP2-knockout HeLa cells reconstituted with EGFP–TP53INP2, we show that TP53INP2 is predominantly degraded by nuclear proteasomes under basal conditions. Under stress, including starvation and various chemical stress inducers, TP53INP2 accumulates in the cytoplasm independently of ATG5, CRM1-mediated export, phosphorylation, ubiquitylation or acetylation. We identify a nuclear localization signal (NLS) overlapping a nucleolar localization signal (NoLS) in the C-terminus, which mediates nuclear import and nucleolar enrichment. Deletion of this region redirects TP53INP2 to LC3B-positive puncta. A conserved nine-amino-acid cytoplasmic retention motif (CRM) in the C-terminus prevents nuclear re-entry under stress. This motif and regulation of subcellular localization is conserved in the related TP53INP1 protein. Fluorescence recovery after photobleaching (FRAP) and importin-binding assays show that nutrient starvation disrupts nuclear import of TP53INP2. Finally, we show that starvation enhances TP53INP2 translation via the m6A demethylase FTO, without altering mRNA stability. These findings uncover coordinated regulation of TP53INP2 localization and turnover by cellular stress.

Ammonia is a natural waste product of cellular metabolism which, through its lysosomotropic ability, can have detrimental effects on various cellular functions. Increased levels of ammonia were recently detected in the interstitial fluid of various tumours, substantiating that high ammonia concentrations are a pathophysiological condition in the tumour microenvironment, alongside hypoxia and acidosis. Since little is known about how cancer cells respond to elevated levels of ammonia in the tumour microenvironment, we investigated how a panel of cancer cell lines derived from solid tumours behaved when exposed to increasing concentrations of ammonia. We found that ammonia represses cell growth, induces genome instability, and inhibits lysosome-mediated proteolysis in a dose-dependent manner. Unexpectedly, we also found that small fluctuations in the pH of the extracellular environment, had a significant impact on the cytotoxic effects of ammonia. In summary, our data suggest that the balance of pH and ammonia within the interstitial fluids of cancerous tumours can have a significant impact on the behaviour and fate of cancer cells.

Macroautophagy/autophagy is a conserved lysosomal degradation process composed of both selective and nonselective degradation pathways. The latter occurs upon nutrient depletion. Selective autophagy exerts quality control of damaged organelles and macromolecules and is going on also under nutrient-replete conditions. Proper regulation of autophagy is vital for cellular homeostasis and prevention of disease. During nutrient availability, autophagy is inhibited by the MTORC1 signaling pathway. However, selective, basal autophagy occurs continuously. How the MTORC1 pathway is fine-tuned to facilitate basal constitutive autophagy is unclear. Recently, we identified the WD-domain repeat protein WDR83/MORG1 as a negative regulator of MTORC1 signaling allowing basal, selective autophagy. WDR83 interacts with both the Ragulator and active RRAG GTPases to prevent recruitment of the MTORC1 complex to the lysosome. Consequently, WDR83 depletion leads to hyperactivation of the MTORC1 pathway and a strong decrease in basal autophagy. As a consequence of WDR83 depletion cell proliferation and migration increase and low levels of WDR83 mRNA are correlated with poor prognosis for several cancers.