bims-proteo 2021-12-05 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2021–12–05
forty papers selected by
Eric Chevet, INSERM

WFS1 functions in ER export of vesicular cargo proteins in pancreatic β-cells.
HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease.
ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function.
Type 1 polyisoprenoid diphosphate phosphatase modulates geranylgeranyl-mediated control of HMG CoA reductase and UBIAD1.
SUMO-SIM interactions: From structure to biological functions.
The E2 ubiquitin-conjugating enzyme HIP2 is a crucial regulator of quality control against mutant SOD1 proteotoxicity.
Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs.
Temporal proteomics reveal specific cell cycle oncoprotein downregulation by p97/VCP inhibition.
ATF6-mediated unfolded protein response facilitates AAV2 transduction by releasing the suppression of AAV receptor on ER stress.
NEDD4L-induced ubiquitination mediating UBE2T degradation inhibits progression of lung adenocarcinoma via PI3K-AKT signaling.
The Role of TRAPγ/SSR3 in Preproinsulin Translocation into the Endoplasmic Reticulum.
MG53 E3 Ligase-dead Mutant Protects Diabetic Hearts from Acute Ischemic/Reperfusion Injury and Ameliorates Diet-induced Cardiometabolic Damage.
pVHL-mediated SMAD3 degradation suppresses TGF-β signaling.
The Spliced Leader RNA Silencing (SLS) Pathway in Trypanosoma brucei Is Induced by Perturbations of Endoplasmic Reticulum, Golgi Complex, or Mitochondrial Protein Factors: Functional Analysis of SLS-Inducing Kinase PK3.
The crystal structure of the FAM134B-GABARAP complex provides mechanistic insights into the selective binding of FAM134 to the GABARAP subfamily.
The RING-domain E3 ubiquitin ligase RNF146 promotes cardiac hypertrophy by suppressing the LKB1/AMPK signaling pathway.
Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery.
The caspase-6-p62 axis modulates p62 droplets based autophagy in a dominant-negative manner.
Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.
The complex interplay between ULK1 and protein phosphatases in autophagy regulation.
Substrate recognition determinants of human eIF2α phosphatases.
Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence.
Cell cycle regulation of ER membrane biogenesis protects against chromosome missegregation.
CRYPTOCHROMES promote daily protein homeostasis.
Fast friends - Ubiquitin-like modifiers as engineered fusion partners.
ASB2 is a novel E3 ligase of SMAD9 required for cardiogenesis.
Peroxisomal support of mitochondrial respiratory efficiency promotes ER stress survival.
Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis.
Alpha-1 antitrypsin deficiency: a re-surfacing adult liver disorder.
GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma.
Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1.
Molecular architecture of the human tRNA ligase complex.
USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1.
Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein.
A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.
A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer.
How multi-component cascades operate in cells: lessons from the ubiquitin system-containing liquid-separated condensates.
O-Glycosylation Induces Amyloid-β To Form New Fibril Polymorphs Vulnerable for Degradation.
Inhibition of cytoplasmic EZH2 induces antitumor activity through stabilization of the DLC1 tumor suppressor protein.
A Guide to Studying Protein Aggregation.

Nat Commun. 2021 Nov 30. 12(1): 6996

WFS1 functions in ER export of vesicular cargo proteins in pancreatic β-cells.

Linlin Wang, Hongyang Liu, Xiaofei Zhang, Eli Song, You Wang, Tao Xu, Zonghong Li.

The sorting of soluble secretory proteins from the endoplasmic reticulum (ER) to the Golgi complex is mediated by coat protein complex II (COPII) vesicles and thought to required specific ER membrane cargo-receptor proteins. However, these receptors remain largely unknown. Herein, we show that ER to Golgi transfer of vesicular cargo proteins requires WFS1, an ER-associated membrane protein whose loss of function leads to Wolfram syndrome. Mechanistically, WFS1 directly binds to vesicular cargo proteins including proinsulin via its ER luminal C-terminal segment, whereas pathogenic mutations within this region disrupt the interaction. The specific ER export signal encoded in the cytosolic N-terminal segment of WFS1 is recognized by the COPII subunit SEC24, generating mature COPII vesicles that traffic to the Golgi complex. WFS1 deficiency leads to abnormal accumulation of proinsulin in the ER, impeding the proinsulin processing as well as insulin secretion. This work identifies a vesicular cargo receptor for ER export and suggests that impaired peptide hormone transport underlies diabetes resulting from pathogenic WFS1 mutations.

DOI: https://doi.org/10.1038/s41467-021-27344-y
Mol Cell. 2021 Nov 23. pii: S1097-2765(21)00948-5. [Epub ahead of print]

HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease.

Juncheng Wei, Bryan T Harada, Dan Lu, Ruihua Ma, Beixue Gao, Yanan Xu, Elena Montauti, Nikita Mani, Shuvam M Chaudhuri, Shana Gregory, Samuel E Weinberg, Donna D Zhang, Richard Green, Chuan He, Deyu Fang.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen triggers an unfolded protein response (UPR) for stress adaptation, the failure of which induces cell apoptosis and tissue/organ damage. The molecular switches underlying how the UPR selects for stress adaptation over apoptosis remain unknown. Here, we discovered that accumulation of unfolded/misfolded proteins selectively induces N6-adenosine-methyltransferase-14 (METTL14) expression. METTL14 promotes C/EBP-homologous protein (CHOP) mRNA decay through its 3' UTR N6-methyladenosine (m6A) to inhibit its downstream pro-apoptotic target gene expression. UPR induces METTL14 expression by competing against the HRD1-ER-associated degradation (ERAD) machinery to block METTL14 ubiquitination and degradation. Therefore, mice with liver-specific METTL14 deletion are highly susceptible to both acute pharmacological and alpha-1 antitrypsin (AAT) deficiency-induced ER proteotoxic stress and liver injury. Further hepatic CHOP deletion protects METTL14 knockout mice from ER-stress-induced liver damage. Our study reveals a crosstalk between ER stress and mRNA m6A modification pathways, termed the ERm6A pathway, for ER stress adaptation to proteotoxicity.

DOI: https://doi.org/10.1016/j.molcel.2021.10.028
Commun Biol. 2021 Dec 02. 4(1): 1350

ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function.

Adrián Cortés Sanchón, Harshitha Santhosh Kumar, Matilde Mantovani, Ivan Osinnii, José María Mateos, Andres Kaech, Dimitri Shcherbakov, Rashid Akbergenov, Erik C Böttger.

Proteostasis is a challenge for cellular organisms, as all known protein synthesis machineries are error-prone. Here we show by cell fractionation and microscopy studies that misfolded proteins formed in the endoplasmic reticulum can become associated with and partly transported into mitochondria, resulting in impaired mitochondrial function. Blocking the endoplasmic reticulum-mitochondria encounter structure (ERMES), but not the mitochondrial sorting and assembly machinery (SAM) or the mitochondrial surveillance pathway components Msp1 and Vms1, abrogated mitochondrial sequestration of ER-misfolded proteins. We term this mitochondria-associated proteostatic mechanism for ER-misfolded proteins ERAMS (ER-associated mitochondrial sequestration). We testify to the relevance of this pathway by using mutant α-1-antitrypsin as an example of a human disease-related misfolded ER protein, and we hypothesize that ERAMS plays a role in pathological features such as mitochondrial dysfunction.

DOI: https://doi.org/10.1038/s42003-021-02873-w
Elife. 2021 Nov 29. pii: e64688. [Epub ahead of print]10

Type 1 polyisoprenoid diphosphate phosphatase modulates geranylgeranyl-mediated control of HMG CoA reductase and UBIAD1.

Rania Elsabrouty, Youngah Jo, Seonghwan Hwang, Dong-Jae Jun, Russell A DeBose-Boyd.

  UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. The prenyltransferase has emerged as a key regulator of sterol-accelerated, endoplasmic reticulum (ER)-associated degradation (ERAD) of HMG CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids including GGpp. Sterols induce binding of UBIAD1 to reductase, inhibiting its ERAD. Geranylgeraniol (GGOH), the alcohol derivative of GGpp, disrupts this binding and thereby stimulates ERAD of reductase and translocation of UBIAD1 to Golgi. We now show that overexpression of Type 1 polyisoprenoid diphosphate phosphatase (PDP1), which dephosphorylates GGpp and other isoprenyl pyrophosphates to corresponding isoprenols, abolishes protein geranylgeranylation as well as GGOH-induced ERAD of reductase and Golgi transport of UBIAD1. Conversely, these reactions are enhanced in the absence of PDP1. Our findings indicate PDP1-mediated hydrolysis of GGpp significantly contributes to a feedback mechanism that maintains optimal intracellular levels of the nonsterol isoprenoid.

Keywords:  ER-associated degradation; biochemistry; cell biology; chemical biology; cholesterol metabolism; human; isoprenoids; prenylation; vesicular transport

DOI:  https://doi.org/10.7554/eLife.64688
Semin Cell Dev Biol. 2021 Nov 25. pii: S1084-9521(21)00283-4. [Epub ahead of print]

SUMO-SIM interactions: From structure to biological functions.

Jara Lascorz, Joan Codina-Fabra, David Reverter, Jordi Torres-Rosell.

  Post-translational modification by Small Ubiquitin-like Modifier (SUMO) proteins regulates numerous cellular processes. This modification involves the covalent and reversible attachment of SUMO to target proteins through an isopeptide bond, using a cascade of E1, E2 and E3 SUMOylation enzymes. Most functions of SUMO depend on the establishment of non-covalent protein-protein interactions between SUMOylated substrates and their binding partners. The vast majority of these interactions involve a conserved surface in the SUMO protein and a SUMO interacting motif (SIM), a short stretch of hydrophobic amino acids and an acidic region, in the interactor protein. Despite single SUMO-SIM interactions are relatively weak, they can have a huge impact at different levels, altering the activity, localization and stability of proteins, triggering the formation of macromolecular assemblies or inducing phase separation. Moreover, SUMO-SIM interactions are ubiquitous in most enzymes of the SUMO pathway, and play essential roles in SUMO conjugation and deconjugation. Here, we analyze the role of SUMO-SIM contacts in SUMO enzymes and targets and discuss how this humble interaction participates in SUMOylation reactions and mediates the outcome of this essential post-translational modification.

Keywords:  E3 ligase; SUMO; SUMO interacting motif; protein-protein interaction; ubiquitin

DOI:  https://doi.org/10.1016/j.semcdb.2021.11.007
Biochim Biophys Acta Mol Basis Dis. 2021 Nov 29. pii: S0925-4439(21)00249-0. [Epub ahead of print] 166316

The E2 ubiquitin-conjugating enzyme HIP2 is a crucial regulator of quality control against mutant SOD1 proteotoxicity.

Yeong Jin Tak, Seongman Kang.

  Mutations in superoxide dismutase 1 (SOD1) leading to the formation of intracellular protein aggregates cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by a selective degeneration of motor neurons. The ALS-linked mutant SOD1 emerged as a possible target for ubiquitin-proteasome system (UPS)-mediated degradation. We aimed to elucidate the role of huntingtin interaction protein 2 (HIP2), an E2 ubiquitin-conjugating enzyme, in the proteotoxicity of mutant SOD1 aggregates. We found that HIP2 interacts with mutant SOD1, but not wild-type SOD1, and is upregulated in response to mutant SOD1 expression. Upregulation of HIP2 protein was observed in the spinal cord of 16-week-old SOD1-G93A transgenic mice. HIP2 further modified mutant SOD1 proteins via K48-linked polyubiquitination and degraded mutant SOD1 proteins through the UPS. Upregulation of HIP2 protected cells from mutant SOD1-induced toxicity. Taken together, our findings demonstrate that HIP2 is a crucial regulator of quality control against the proteotoxicity of mutant SOD1. Our results suggest that modulating HIP2 may represent a novel therapeutic strategy for the treatment of ALS.

Keywords:  ALS; HIP2; Protein aggregates; Proteotoxicity; SOD1; UPS

DOI:  https://doi.org/10.1016/j.bbadis.2021.166316
Nat Commun. 2021 Dec 01. 12(1): 7013

Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs.

Nathalia Varejão, Jara Lascorz, Joan Codina-Fabra, Gemma Bellí, Helena Borràs-Gas, Jordi Torres-Rosell, David Reverter.

Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. Smc5/6 is a nuclear multi-subunit complex that participates in the recombinational DNA repair processes and is required in the maintenance of chromosome integrity. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester for discharge on the substrate. Here we present the crystal structure of the SUMO E3 ligase Nse2 in complex with an E2-SUMO thioester mimetic. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse2 are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction. Both SIM interfaces are essential in the activity of Nse2 and are required to cope with DNA damage.

DOI: https://doi.org/10.1038/s41467-021-27301-9
Cell Chem Biol. 2021 Nov 23. pii: S2451-9456(21)00482-7. [Epub ahead of print]

Temporal proteomics reveal specific cell cycle oncoprotein downregulation by p97/VCP inhibition.

Feng Wang, Shan Li, Nadia Houerbi, Tsui-Fen Chou.

  Targeting protein quality control (PQC) pathways using proteasome or p97/VCP inhibition can effectively treat blood tumors. However, in solid tumors, only p97/VCP inhibitors are effective. To probe this difference in efficacy, we tracked HCT116 colon cancer cells using temporal proteomics to define the cellular and molecular responses to proteasome and p97 inhibition. Proteins involved in general PQC pathways were similarly upregulated by both treatments, suggesting that the proteotoxic stress caused by inhibitors does not explain the differential therapeutic effectiveness. Unexpectedly, proteins specifically dysregulated by two p97 inhibitors are involved in cell cycle control. Indeed, eleven cell cycle proteins were downregulated by p97 inhibition but not by proteasome inhibition. Western blot analysis validated the degradation of cyclin D1 and Securin, which depends on proteasome but not on p97. Differing regulation of cell cycle proteins by p97 and the proteasome may, therefore, explain the therapeutic efficacy of p97 inhibitors in colon cancer.

Keywords:  CB-5083; E2F1; NMS-873; UPCDC-30245; anticancer; cell cycle; cyclin D1; p97 inhibitor; proteasome inhibitor; proteomic

DOI:  https://doi.org/10.1016/j.chembiol.2021.11.005
J Virol. 2021 Dec 01. JVI0110321

ATF6-mediated unfolded protein response facilitates AAV2 transduction by releasing the suppression of AAV receptor on ER stress.

Mengtian Cui, Qingfang Zhao, Jing Wang, Yang Si, Shan Cheng, Wei Ding.

Adeno-associated virus (AAV) is extensively used as a viral vector to deliver therapeutic genes during human gene therapy. A high affinity cellular receptor (AAVR) for most serotypes was recently identified, however, its biological function as a gene product remains unclear. In this study, we used AAVR knockdown cell models to show that AAVR depletion significantly attenuated cells to activate unfolded protein response (UPR) pathways, when exposed to the endoplasmic reticulum (ER) stress inducer, tunicamycin. By analyzing three major UPR pathways, we found that ATF6 signaling was most affected in an AAVR-dependent fashion, distinct to CHOP and XBP1 branches. AAVR capacity in UPR regulation required the full native AAVR protein, and AAV2 capsid binding to the receptor altered ATF6 dynamics. Conversely, the transduction efficiency of AAV2 was associated with changes in ATF6 signaling in host cells following treatment with different small molecules. Thus, AAVR served as an inhibitory molecule to repress UPR responses via a specificity for ATF6 signaling, and the AAV2 infection route involved the release from AAVR-mediated ATF6 repression, thereby facilitating viral intracellular trafficking and transduction. Importance The native function of the AAVR as an ER-Golgi localized protein is largely unknown. We showed that AAVR acted as a functional molecule to regulate UPR signaling under induced ER stress. AAVR inhibited the activation of the transcription factor, ATF6, whereas receptor binding to AAV2 released the suppression effects. This finding has expanded our understanding of AAV infection biology in terms of the physiological properties of AAVR in host cells. Importantly, our research provides a possible strategy which may improve the efficiency of AAV mediated gene delivery during gene therapy.

DOI: https://doi.org/10.1128/JVI.01103-21
Cancer Cell Int. 2021 Nov 27. 21(1): 631

NEDD4L-induced ubiquitination mediating UBE2T degradation inhibits progression of lung adenocarcinoma via PI3K-AKT signaling.

Yongbing Chen, Haihua Hong, Qingqing Wang, Junqiang Li, Wenfeng Zhang, Tingting Chen, Pu Li.

   BACKGROUND: A number of studies have indicated that Ubiquitin-conjugating enzyme E2T (UBE2T), as an oncogene, promotes progression and metastasis of lung cancer, including lung adenocarcinoma (LUAD), but it is completely unknown whether and how UBE2T is ubiquitylated and degraded, and by which E3 ligase. NEDD4L plays a critical role in the regulation of cellular processes of various cancers, most of which is attributed to its E3 ubiquitin ligase function. However, the relationship between NEDD4L and UBE2T in LUAD has not been elucidated.
METHODS: The relationship between NEDD4L and UBE2T in LUAD tissues and cells was found by bioinformatic analyses and immunoblotting. Cell counting kit-8, colony formation assay, half-life analysis and the in vivo ubiquitylation assay, generation of xenograft model were performed to determine how NEDD4L regulates UBE2T and its downstream signaling pathway in vitro and in vivo.
RESULTS: Bioinformatic analyses found that NEDD4L, as a potential correlation E3 ligase of UBE2T, was negatively correlated with UBE2T in LUAD. Consistently, UBE2T protein half-life was shortened or extended by NEDD4L overexpression or depletion, respectively. NEDD4L inhibited LUAD cell progression in vitro and in vivo via inducing the ubiquitination-mediated UBE2T degradation, which repressed PI3K-AKT signaling. Similarly, NEDD4L predicted a better patient survival, whereas UBE2T predicted a worse survival.
CONCLUSIONS: Collectively, our results reveal that NEDD4L is a novel E3 ligase of UBE2T, which can inhibit PI3K-AKT signaling by targeting for UBE2T ubiquitination and degradation, resulting in repression of LUAD cell progression.

Keywords:  Lung adenocarcinoma; NEDD4L; PI3K-AKT signaling; UBE2T; Ubiquitylation

DOI:  https://doi.org/10.1186/s12935-021-02341-9
Diabetes. 2021 Dec 02. pii: db210638. [Epub ahead of print]

The Role of TRAPγ/SSR3 in Preproinsulin Translocation into the Endoplasmic Reticulum.

Xiaoxi Xu, Yumeng Huang, Xin Li, Peter Arvan, Ming Liu.

In the endoplasmic reticulum (ER), the Translocation-Associated Protein complex (TRAP, also called Signal sequence receptor, SSR) includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2 and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2 or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting co-translational and post-translational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels - observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkable, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis.

DOI: https://doi.org/10.2337/db21-0638
Diabetes. 2021 Nov 29. pii: db210322. [Epub ahead of print]

MG53 E3 Ligase-dead Mutant Protects Diabetic Hearts from Acute Ischemic/Reperfusion Injury and Ameliorates Diet-induced Cardiometabolic Damage.

Han Feng, Hao Shen, Matthew J Robeson, Yue-Han Wu, Wu Hong-Kun, Geng-Jia Chen, Shuo Zhang, Peng Xie, Li Jin, Yanyun He, Yingfan Wang, Fengxiang Lv, Xinli Hu, Yan Zhang, Xiao Rui-Ping.

Cardiometabolic diseases, including diabetes and its cardiovascular complications, are the global leading cause of death, highlighting a major unmet medical need. Over the last decade, MG53, also named TRIM72, has emerged as a powerful agent for myocardial membrane repair and cardioprotection, but its therapeutic value is complicated by its E3 ligase activity that mediates metabolic disorders. Here, we show that an E3 ligase-dead mutant, MG53-C14A, retains its cardioprotective function without causing metabolic side-effects. When administrated in normal animals, both recombinant human wild type MG53 protein (rhMG53-WT) and its E3 ligase-dead mutant (rhMG53-C14A) protect the heart equally from myocardial infarction and ischemia/reperfusion (I/R) injury. However, in diabetic db/db mice, rhMG53-WT treatment markedly aggravates hyperglycemia, cardiac I/R injury and mortality, whereas acute and chronic treatment of rhMG53-C14A still effectively ameliorates I/R-induced myocardial injury and mortality or diabetic cardiomyopathy, respectively, without inflicting metabolic side-effects. Furthermore, knock-in of MG53-C14A protects the mice from high-fat diet-induced metabolic disorders and cardiac damage. Thus, the E3 ligase-dead mutant MG53-C14A not only protects the heart from acute myocardial injury but also counteract metabolic stress, providing a potentially important therapy for the treatment of acute myocardial injuries amidst metabolic disorders, including diabetes and obesity.

DOI: https://doi.org/10.2337/db21-0322
J Cell Biol. 2022 Jan 03. pii: e202012097. [Epub ahead of print]221(1):

pVHL-mediated SMAD3 degradation suppresses TGF-β signaling.

Jun Zhou, Yasamin Dabiri, Rodrigo A Gama-Brambila, Shahrouz Ghafoory, Mukaddes Altinbay, Arianeb Mehrabi, Mohammad Golriz, Biljana Blagojevic, Stefanie Reuter, Kang Han, Anna Seidel, Ivan Đikić, Stefan Wölfl, Xinlai Cheng.

Transforming growth factor β (TGF-β) signaling plays a fundamental role in metazoan development and tissue homeostasis. However, the molecular mechanisms concerning the ubiquitin-related dynamic regulation of TGF-β signaling are not thoroughly understood. Using a combination of proteomics and an siRNA screen, we identify pVHL as an E3 ligase for SMAD3 ubiquitination. We show that pVHL directly interacts with conserved lysine and proline residues in the MH2 domain of SMAD3, triggering degradation. As a result, the level of pVHL expression negatively correlates with the expression and activity of SMAD3 in cells, Drosophila wing, and patient tissues. In Drosophila, loss of pVHL leads to the up-regulation of TGF-β targets visible in a downward wing blade phenotype, which is rescued by inhibition of SMAD activity. Drosophila pVHL expression exhibited ectopic veinlets and reduced wing growth in a similar manner as upon loss of TGF-β/SMAD signaling. Thus, our study demonstrates a conserved role of pVHL in the regulation of TGF-β/SMAD3 signaling in human cells and Drosophila wing development.

DOI: https://doi.org/10.1083/jcb.202012097
mBio. 2021 Nov 30. e0260221

The Spliced Leader RNA Silencing (SLS) Pathway in Trypanosoma brucei Is Induced by Perturbations of Endoplasmic Reticulum, Golgi Complex, or Mitochondrial Protein Factors: Functional Analysis of SLS-Inducing Kinase PK3.

Uthman Okalang, Bar Mualem Bar-Ner, K Shanmugha Rajan, Nehemya Friedman, Saurav Aryal, Katarina Egarmina, Ronen Hope, Netaly Khazanov, Hanoch Senderowitz, Assaf Alon, Deborah Fass, Shulamit Michaeli.

  In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5' exon derived from the spliced leader (SL) RNA. Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA-binding protein TRF4, leading to the shutoff of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structures of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all of the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments. IMPORTANCE In this study, we found that SLS is induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. We also report on the autophosphorylation of PK3 during SLS induction. This study has implications for our understanding of how trypanosomes keep the homeostasis between the ER and the mitochondria and suggests that PK3 may participate in the connection between these two organelles. The pathway, when induced, leads to the suicide of these parasites, and its induction offers a potential novel drug target against these parasites.

Keywords:  BiP; ERO1; PK3-PERK homologue; QSOX; SLS; TIMRHOM1; calreticulin; spliced leader RNA silencing; trypanosomes

DOI:  https://doi.org/10.1128/mBio.02602-21
FEBS Open Bio. 2021 Dec 01.

The crystal structure of the FAM134B-GABARAP complex provides mechanistic insights into the selective binding of FAM134 to the GABARAP subfamily.

Junfeng Zhao, Zhiwei Li, Jianchao Li.

  The mammalian Atg8 family (Atg8s proteins) consists of two subfamilies: GABARAP and LC3. All members can bind to the LC3-interacting region (LIR) or Atg8-interacting motif (AIM) and participate in multiple steps of autophagy. The endoplasmic reticulum (ER) autophagy receptor FAM134B contains an LIR motif that can bind to Atg8s, but whether it can differentially bind to the two subfamilies and, if so, the structural basis for this preference remain unknown. Here we found that FAM134B bound to the GABARAP subfamily more strongly than to the LC3 subfamily. We then solved the crystal structure of the FAM134B-GABARAP complex and demonstrated that FAM134B used both its LIR core and the C-terminal helix to bind to GABARAP. We further showed that these properties might be conserved in FAM134A or FAM134C. The structure also allowed us to identify the structural determinants for the binding selectivity. Our work may be valuable for studying the differential functions of GABARAP and LC3 subfamilies in ER-phagy in the future.

Keywords:  Atg8; Autophagy; FAM134B; GABARAP; LC3 interacting region/Atg8 interacting motif

DOI:  https://doi.org/10.1002/2211-5463.13340
Exp Cell Res. 2021 Nov 29. pii: S0014-4827(21)00510-3. [Epub ahead of print] 112954

The RING-domain E3 ubiquitin ligase RNF146 promotes cardiac hypertrophy by suppressing the LKB1/AMPK signaling pathway.

Zhiyong Sheng, Jianning Xu, Fuxing Li, Ying Yuan, Xiaogang Peng, Shenjian Chen, Rui Zhou, Wei Huang.

  The RING-domain E3 ubiquitin ligase RNF146 is an enzyme that plays an important role in ubiquitin-proteasomal protein degradation and participates in various pathophysiological processes. However, its role in cardiac hypertrophy is unclear. In the present work, thoracic transverse aortic constriction (TAC) was performed in transgenic mice with RNF146 knockout mice (KO) and wild-type mice, and neonatal rat cardiomyocytes (NRCMs) were subjected to angiotensin II (Ang II) stimulation to induce cardiac hypertrophy in vitro and in vivo. RNF146 expression was significantly increased in hypertrophied murine hearts and Ang II-stimulated NRCMs. RNF146-KO mice and knockdown of RNF146 NRCMs attenuated TAC- or Ang II-stimulated cardiac hypertrophy. Conversely, enforced expression of RNF146 aggravated these changes. Mechanistically, we found that RNF146 KO or knockdown increased the activation of the AMP-activated protein kinase (AMPK) pathway. Furthermore, we found that RNF146 KO or knockdown decreased ubiquitination of Liver kinase B1 (LKB1), which promoted the activation of the AMPK pathway in a dependent manner. In conclusion, RNF146 targets LKB1 protein for ubiquitin-proteasome degradation in cardiomyocytes and subsequently promotes cardiac hypertrophy by suppressing the activation of the AMPK signaling pathway.

Keywords:  AMPK; Cardiac hypertrophy; LKB1; RNF146; Ubiquitination

DOI:  https://doi.org/10.1016/j.yexcr.2021.112954
Mol Cell. 2021 Nov 19. pii: S1097-2765(21)00954-0. [Epub ahead of print]

Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery.

Jasmin Adriana Schäfer, Süleyman Bozkurt, Jonas Benjamin Michaelis, Kevin Klann, Christian Münch.

  Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human pathologies. However, a lack of suitable tools to measure protein uptake across the mitochondrial proteome has prevented the identification of specific proteins affected by import perturbation. Here, we introduce mePRODmt, a pulsed-SILAC based proteomics approach that includes a booster signal to increase the sensitivity for mitochondrial proteins selectively, enabling global dynamic analysis of endogenous mitochondrial protein uptake in cells. We applied mePRODmt to determine protein uptake kinetics and examined how inhibitors of mitochondrial import machineries affect protein uptake. Monitoring changes in translation and uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated by the import and translation machineries via activation of the integrated stress response. Strikingly, uptake changes were not uniform, with subsets of proteins being unaffected or decreased due to changes in translation or import capacity.

Keywords:  SILAC; TMT; disease; integrated stress response; mitochondria; protein translocation; proteomics; proteostasis; respiratory chain complexes; translation

DOI:  https://doi.org/10.1016/j.molcel.2021.11.004
Cell Death Differ. 2021 Dec 03.

The caspase-6-p62 axis modulates p62 droplets based autophagy in a dominant-negative manner.

Evelina Valionyte, Yi Yang, Sophie A Griffiths, Amelia T Bone, Elizabeth R Barrow, Vikram Sharma, Boxun Lu, Shouqing Luo.

SQSTM1/p62, as a major autophagy receptor, forms droplets that are critical for cargo recognition, nucleation, and clearance. p62 droplets also function as liquid assembly platforms to allow the formation of autophagosomes at their surfaces. It is unknown how p62-droplet formation is regulated under physiological or pathological conditions. Here, we report that p62-droplet formation is selectively blocked by inflammatory toxicity, which induces cleavage of p62 by caspase-6 at a novel cleavage site D256, a conserved site across human, mouse, rat, and zebrafish. The N-terminal cleavage product is relatively stable, whereas the C-terminal product appears undetectable. Using a variety of cellular models, we show that the p62 N-terminal caspase-6 cleavage product (p62-N) plays a dominant-negative role to block p62-droplet formation. In vitro p62 phase separation assays confirm this observation. Dominant-negative regulation of p62-droplet formation by caspase-6 cleavage attenuates p62 droplets dependent autophagosome formation. Our study suggests a novel pathway to modulate autophagy through the caspase-6-p62 axis under certain stress stimuli.

DOI: https://doi.org/10.1038/s41418-021-00912-x
Nucleic Acids Res. 2021 Dec 01. pii: gkab1174. [Epub ahead of print]

Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.

Haixia Zhou, Manal S Zaher, Johannes C Walter, Alan Brown.

When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.

DOI: https://doi.org/10.1093/nar/gkab1174
Autophagy. 2021 Nov 28. 1-2

The complex interplay between ULK1 and protein phosphatases in autophagy regulation.

Devanarayanan Siva Sankar, Zehan Hu, Joern Dengjel.

  ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome biogenesis. To uncover if ULK1 has broader functions also regulating subsequent steps of autophagosome turnover, i.e., maturation, lysosomal fusion, and degradation, we performed a set of unbiased phosphoproteomic experiments employing mouse and human cells in combination with genetic and environmental perturbations. We characterized more than 1,000 potential ULK1 target sites of which many affect proteins known to be involved in all phases of the autophagosome life cycle. To better understand which of these 1,000 phosphosites were directly phosphorylated by ULK1, in contrast to downstream kinases being activated or phosphatases being inhibited by ULK1, we developed a proteome-scale in vitro kinase assay and characterized 187 phosphosites on 157 proteins as bona fide ULK1 target sites. Interestingly, our results highlight an intricate crosstalk between ULK1 and protein phosphatases. Focusing on STRN (striatin), a regulatory subunit of PPP2/PP2A (protein phosphatase 2), we identified a positive feedback loop linked to ULK1 and promoting autophagy.

Keywords:  Feedback; STRIPAK; STRN; ULK1; in vitro kinase assay; kinase; phosphatase; phosphoproteomics; phosphorylation; striatin

DOI:  https://doi.org/10.1080/15548627.2021.2002546
Open Biol. 2021 Dec;11(12): 210205

Substrate recognition determinants of human eIF2α phosphatases.

George Hodgson, Antonina Andreeva, Anne Bertolotti.

  Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Here, we generated a series of R15 truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. G-actin, which has been proposed to confer selectivity to R15 phosphatases, does not bind these regions, indicating that it is not required for substrate binding. Fragments containing the substrate-binding regions but lacking the PP1-binding motif trapped the phospho-substrate and caused accumulation of phosphorylated eIF2α in unstressed cells. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1-binding region, exhibit wild-type activity. This work identifies the substrate-binding region in R15s, that functions as a phospho-substrate trapping mutant, thereby defining a key region of R15s for follow up studies.

Keywords:  PP1; PPP1R15; eIF2α phosphorylation; integrated stress response; phosphatase

DOI:  https://doi.org/10.1098/rsob.210205
FEBS J. 2021 Nov 28.

Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence.

Helena Zábranská, Aleš Zábranský, Barbora Lubyová, Jan Hodek, Alena Křenková, Martin Hubálek, Jan Weber, Iva Pichová.

  Hepatitis B virus (HBV) uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex (TRAP). We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.

Keywords:  ER translocation; HBV precore protein; HBe; Hepatitis B virus; TRAP complex; cysteine residues; signal peptide

DOI:  https://doi.org/10.1111/febs.16304
Dev Cell. 2021 Nov 24. pii: S1534-5807(21)00894-7. [Epub ahead of print]

Cell cycle regulation of ER membrane biogenesis protects against chromosome missegregation.

Holly Merta, Jake W Carrasquillo Rodríguez, Maya I Anjur-Dietrich, Tevis Vitale, Mitchell E Granade, Thurl E Harris, Daniel J Needleman, Shirin Bahmanyar.

  Failure to reorganize the endoplasmic reticulum (ER) in mitosis results in chromosome missegregation. Here, we show that accurate chromosome segregation in human cells requires cell cycle-regulated ER membrane production. Excess ER membranes increase the viscosity of the mitotic cytoplasm to physically restrict chromosome movements, which impedes the correction of mitotic errors leading to the formation of micronuclei. Mechanistically, we demonstrate that the protein phosphatase CTDNEP1 counteracts mTOR kinase to establish a dephosphorylated pool of the phosphatidic acid phosphatase lipin 1 in interphase. CTDNEP1 control of lipin 1 limits the synthesis of fatty acids for ER membrane biogenesis in interphase that then protects against chromosome missegregation in mitosis. Thus, regulation of ER size can dictate the biophysical properties of mitotic cells, providing an explanation for why ER reorganization is necessary for mitotic fidelity. Our data further suggest that dysregulated lipid metabolism is a potential source of aneuploidy in cancer cells.

Keywords:  CTDNEP1; aneuploidy; lipid homeostasis; lipin; mTOR; medulloblastoma; micronuclei; mitosis; nuclear assembly

DOI:  https://doi.org/10.1016/j.devcel.2021.11.009
EMBO J. 2021 Nov 29. e2021108883

CRYPTOCHROMES promote daily protein homeostasis.

David C S Wong, Estere Seinkmane, Aiwei Zeng, Alessandra Stangherlin, Nina M Rzechorzek, Andrew D Beale, Jason Day, Martin Reed, Sew Y Peak-Chew, Christine T Styles, Rachel S Edgar, Marrit Putker, John S O'Neill.

  The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Keywords:  CRYPTOCHROME; circadian rhythm; clock mutant; protein homeostasis; proteotoxic stress

DOI:  https://doi.org/10.15252/embj.2021108883
Semin Cell Dev Biol. 2021 Nov 25. pii: S1084-9521(21)00289-5. [Epub ahead of print]

Fast friends - Ubiquitin-like modifiers as engineered fusion partners.

Evrydiki Asimaki, Kirill Petriukov, Christian Renz, Cindy Meister, Helle D Ulrich.

  Ubiquitin and its relatives are major players in many biological pathways, and a variety of experimental tools based on biological chemistry or protein engineering is available for their manipulation. One popular approach is the use of linear fusions between the modifier and a protein of interest. Such artificial constructs can facilitate the understanding of the role of ubiquitin in biological processes and can be exploited to control protein stability, interactions and degradation. Here we summarize the basic design considerations and discuss the advantages as well as limitations associated with their use. Finally, we will refer to several published case studies highlighting the principles of how they provide insight into pathways ranging from membrane protein trafficking to the control of epigenetic modifications.

Keywords:  Degron technology; Endocytosis; Genome maintenance; Mitophagy; Protein engineering; Protein stability; SUMO; Transcriptional regulation; UFD; Ubiquitin

DOI:  https://doi.org/10.1016/j.semcdb.2021.11.013
Sci Rep. 2021 Nov 29. 11(1): 23056

ASB2 is a novel E3 ligase of SMAD9 required for cardiogenesis.

Kyung-Duk Min, Masanori Asakura, Manabu Shirai, Satoru Yamazaki, Shin Ito, Hai Ying Fu, Hiroshi Asanuma, Yoshihiro Asano, Tetsuo Minamino, Seiji Takashima, Masafumi Kitakaze.

Cardiogenesis requires the orchestrated spatiotemporal tuning of BMP signalling upon the balance between induction and counter-acting suppression of the differentiation of the cardiac tissue. SMADs are key intracellular transducers and the selective degradation of SMADs by the ubiquitin-proteasome system is pivotal in the spatiotemporal tuning of BMP signalling. However, among three SMADs for BMP signalling, SMAD1/5/9, only the specific E3 ligase of SMAD9 remains poorly investigated. Here, we report for the first time that SMAD9, but not the other SMADs, is ubiquitylated by the E3 ligase ASB2 and targeted for proteasomal degradation. ASB2, as well as Smad9, is conserved among vertebrates. ASB2 expression was specific to the cardiac region from the very early stage of cardiac differentiation in embryogenesis of mouse. Knockdown of Asb2 in zebrafish resulted in a thinned ventricular wall and dilated ventricle, which were rescued by simultaneous knockdown of Smad9. Abundant Smad9 protein leads to dysregulated cardiac differentiation through a mechanism involving Tbx2, and the BMP signal conducted by Smad9 was downregulated under quantitative suppression of Smad9 by Asb2. Our findings demonstrate that ASB2 is the E3 ligase of SMAD9 and plays a pivotal role in cardiogenesis through regulating BMP signalling.

DOI: https://doi.org/10.1038/s41598-021-02390-0
J Cell Sci. 2021 Dec 02. pii: jcs.259254. [Epub ahead of print]

Peroxisomal support of mitochondrial respiratory efficiency promotes ER stress survival.

Imadeddin Hijazi, Emily Wang, Michelle Orozco, Sarah Pelton, Amy Chang.

  Endoplasmic reticulum stress (ERS) occurs when cellular demand for protein folding exceeds the capacity of the organelle. Adaptation and cell survival in response to ERS requires a critical contribution by mitochondria and peroxisomes. During ERS response, mitochondrial respiration increases to ameliorate reactive oxygen species (ROS) accumulation; we now show in yeast that peroxisome abundance also increases to promote an adaptive response. In pox1▵ cells, defective in peroxisomal ß oxidation of fatty acids, respiratory response to ERS is impaired, and ROS accrues. However, respiratory response to ERS is rescued, and ROS production is mitigated in pox1▵ cells by overexpression of Mpc1, the mitochondrial pyruvate carrier that provides another source of acetyl CoA to fuel the TCA cycle and oxidative phosphorylation. Using proteomics, select mitochondrial proteins were identified that undergo upregulation by ERS to remodel respiratory machinery. Several peroxisome-based proteins were also increased, corroborating the peroxisomal role in ERS adaptation. Finally, ERS stimulates assembly of respiratory complexes into higher order supercomplexes, underlying increased electron transfer efficiency. Our results highlight peroxisomal and mitochondrial support for ERS adaptation to favor cell survival.

Keywords:  Endoplasmic reticulum; Mitochondria; Stress survival

DOI:  https://doi.org/10.1242/jcs.259254
Nat Commun. 2021 Nov 30. 12(1): 6984

Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis.

Maxime Uriarte, Nadine Sen Nkwe, Roch Tremblay, Oumaima Ahmed, Clémence Messmer, Nazar Mashtalir, Haithem Barbour, Louis Masclef, Marion Voide, Claire Viallard, Salima Daou, Djaileb Abdelhadi, Daryl Ronato, Mohammadjavad Paydar, Anaïs Darracq, Karine Boulay, Nicolas Desjardins-Lecavalier, Przemyslaw Sapieha, Jean-Yves Masson, Mikhail Sergeev, Benjamin H Kwok, Laura Hulea, Frédérick A Mallette, Eric Milot, Bruno Larrivée, Hugo Wurtele, El Bachir Affar.

Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions.

DOI: https://doi.org/10.1038/s41467-021-27306-4
J Hepatol. 2021 Nov 27. pii: S0168-8278(21)02227-3. [Epub ahead of print]

Alpha-1 antitrypsin deficiency: a re-surfacing adult liver disorder.

Malin Fromme, Carolin V Schneider, Christian Trautwein, Nicola Brunetti-Pierri, Pavel Strnad.

  Alpha-1 antitrypsin deficiency (AATD) arises from mutations in the SERPINA1 gene encoding alpha-1 antitrypsin (AAT) that lead to AAT retention in the endoplasmic reticulum of hepatocytes, causing proteotoxic liver injury and loss-of-function lung disease. The homozygous Pi*Z mutation (Pi*ZZ genotype) is responsible for the majority of severe AATD cases and can precipitate both pediatric and adult liver diseases, while the heterozygous Pi*Z mutation (Pi*MZ genotype) is an established modifier of liver disease. We review genotype-related hepatic phenotypes/disease predispositions. We also describe the mechanisms and factors promoting the development of liver disease, as well as approaches to evaluate the extent of liver fibrosis. We discuss the emerging therapeutic approaches, diagnosis, and clinical management of this neglected disorder.

Keywords:  Fibroscan; Pi*S; Pi*Z; SERPINA1; liver cirrhosis; liver fibrosis

DOI:  https://doi.org/10.1016/j.jhep.2021.11.022
Clin Transl Med. 2021 11;11(11): e635

GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma.

Qian Yun Ge, Jin Chen, Gan Xun Li, Xiao Long Tan, Jia Song, Deng Ning, Jie Mo, Peng Cheng Du, Qiu Meng Liu, Hui Fang Liang, Ze Yang Ding, Xue Wu Zhang, Bi Xiang Zhang.

   BACKGROUND: Aberrant TAK1 (transforming growth factor β-activated kinase 1) activity is known to be involved in a variety of malignancies, but the regulatory mechanisms of TAK1 remain poorly understood. GRAMD4 (glucosyltransferase Rab-like GTPase activator and myotubularin domain containing 4) is a newly discovered p53-independent proapoptotic protein with an unclear role in HCC (hepatocellular carcinoma).
RESULTS: In this research, we found that GRAMD4 expression was lower in HCC samples, and its downregulation predicted worse prognosis for patients after surgical resection. Functionally, GRAMD4 inhibited HCC migration, invasion and metastasis. Mechanistically, GRAMD4 interacted with TAK1 to promote its protein degradation, thus, resulting in the inactivation of MAPK (Mitogen-activated protein kinase) and NF-κB pathways. Furthermore, GRAMD4 was proved to recruit ITCH (itchy E3 ubiquitin protein ligase) to promote the ubiquitination of TAK1. Moreover, high expression of TAK1 was correlated with low expression of GRAMD4 in HCC patients.
CONCLUSIONS: GRAMD4 inhibits the migration and metastasis of HCC, mainly by recruiting ITCH to promote the degradation of TAK1, which leads to the inactivation of MAPK and NF-κB signalling pathways.

Keywords:  GRAMD4; HCC; Metastasis; TAK1; Ubiquitination

DOI:  https://doi.org/10.1002/ctm2.635
J Extracell Vesicles. 2021 Dec;10(14): e12166

Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1.

Tom G Keulers, Sten F Libregts, Joel E J Beaumont, Kim G Savelkouls, Johan Bussink, Hans Duimel, Ludwig Dubois, Marijke I Zonneveld, Carmen López-Iglesias, Karel Bezstarosti, Jeroen A Demmers, Marc Vooijs, Marca Wauben, Kasper M A Rouschop.

  Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

Keywords:  GABARAPL1; autophagy; exosomes; extracellular vesicles; hypoxia

DOI:  https://doi.org/10.1002/jev2.12166
Elife. 2021 Dec 02. pii: e71656. [Epub ahead of print]10

Molecular architecture of the human tRNA ligase complex.

Alena Kroupova, Fabian Ackle, Igor Asanović, Stefan Weitzer, Franziska M Boneberg, Marco Faini, Alexander Leitner, Alessia Chui, Ruedi Aebersold, Javier Martinez, Martin Jinek.

  RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.

Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.71656
Cell Death Differ. 2021 Nov 27.

USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1.

Yuluo Rong, Jin Fan, Chengyue Ji, Zhuanghui Wang, Xuhui Ge, Jiaxing Wang, Wu Ye, Guoyong Yin, Weihua Cai, Wei Liu.

Spinal cord ischemia-reperfusion injury (SCIRI) is a serious trauma that can lead to loss of sensory and motor function. Ferroptosis is a new form of regulatory cell death characterized by iron-dependent accumulation of lipid peroxides. Ferroptosis has been studied in various diseases; however, the exact function and molecular mechanism of ferroptosis in SCIRI remain unknown. In this study, we demonstrated that ferroptosis is involved in the pathological mechanism of SCIRI. Inhibition of ferroptosis could promote the recovery of motor function in mice after SCIRI. In addition, we found that ubiquitin-specific protease 11 (USP11) was significantly upregulated in neuronal cells after hypoxia-reoxygenation and in the spinal cord in mice with I/R injury. Knockdown of USP11 in vitro and KO of USP11 in vivo (USP11-/Y) significantly decreased neuronal cell ferroptosis. In mice, this promotes functional recovery after SCIRI. In contrast, in vitro, USP11 overexpression leads to classic ferroptosis events. Overexpression of USP11 in mice resulted in increased ferroptosis and poor functional recovery after SCIRI. Interestingly, upregulating the expression of USP11 also appeared to increase the production of autophagosomes and to cause substantial autophagic flux, a potential mechanism through which USP11 may enhance ferroptosis. The decreased autophagy markedly weakened the ferroptosis mediated by USP11 and autophagy induction had a synergistic effect with USP11. Importantly, USP11 promotes autophagy activation by stabilizing Beclin 1, thereby leading to ferroptosis. In conclusion, this study shows that ferroptosis is closely associated with SCIRI, and that USP11 plays a key role in regulating ferroptosis and additionally identifies USP11-mediated autophagy-dependent ferroptosis as a promising target for the treatment of SCIRI.

DOI: https://doi.org/10.1038/s41418-021-00907-8
Genetics. 2021 Oct 02. pii: iyab129. [Epub ahead of print]219(2):

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein.

Jyotsna Kumar, Michael Reidy, Daniel C Masison.

  [PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.

Keywords:  J-protein; Sis1; Sup35; amyloid; chaperone; prion; proteostasis

DOI:  https://doi.org/10.1093/genetics/iyab129
PLoS Pathog. 2021 Nov 29. 17(11): e1009409

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.

Charlotte A Stoneham, Simon Langer, Paul D De Jesus, Jacob M Wozniak, John Lapek, Thomas Deerinck, Andrea Thor, Lars Pache, Sumit K Chanda, David J Gonzalez, Mark Ellisman, John Guatelli.

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

DOI: https://doi.org/10.1371/journal.ppat.1009409
Bioorg Chem. 2021 Nov 20. pii: S0045-2068(21)00883-X. [Epub ahead of print] 105505

A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer.

Svenja Lier, Andreas Sellmer, Felix Orben, Stephanie Heinzlmeir, Lukas Krauß, Christian Schneeweis, Zonera Hassan, Carolin Schneider, Arlette Schäfer, Herwig Pongratz, Thomas Engleitner, Rupert Öllinger, Anna Kuisl, Florian Bassermann, Christoph Schlag, Bo Kong, Stefan Dove, Bernhard Kuster, Roland Rad, Maximilian Reichert, Matthias Wirth, Dieter Saur, Siavosh Mahboobi, Günter Schneider.

  Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.

Keywords:  Cereblon; GSPT1; GSPT2; MYC; PLK1

DOI:  https://doi.org/10.1016/j.bioorg.2021.105505
Mol Cell Oncol. 2021 ;8(5): 1989939

How multi-component cascades operate in cells: lessons from the ubiquitin system-containing liquid-separated condensates.

Afu Fu, Ido Livneh, Aaron Ciechanover, Victoria Cohen-Kaplan.

  Membraneless condensates have recently caught the attention of biologists as hubs for cellular components required for catalysis of basic processes. Whether they are real has become the center of heated discussion where the main issues are their mechanism of assembly and function. A recent study describing these condensates as hubs for protein degradation by the ubiquitin system may shed a new light on this recent development in cell biology.

Keywords:  LLPS condensates; p62; proteasome; protein degradation; ubiquitin

DOI:  https://doi.org/10.1080/23723556.2021.1989939
J Am Chem Soc. 2021 Nov 29.

O-Glycosylation Induces Amyloid-β To Form New Fibril Polymorphs Vulnerable for Degradation.

Dangliang Liu, Qijia Wei, Wencheng Xia, Changdong He, Qikai Zhang, Lu Huang, Xiaoya Wang, Yunpeng Sun, Yeyang Ma, Xiaohui Zhang, Yuan Wang, Xiaomeng Shi, Cong Liu, Suwei Dong.

Brain accumulation of amyloid-β (Aβ) peptides (resulting from a disrupted balance between biosynthesis and clearance) occurs during the progression of Alzheimer's disease (AD). Aβ peptides have diverse posttranslational modifications (PTMs) that variously modulate Aβ aggregation into fibrils, but understanding the mechanistic roles of PTMs in these processes remains a challenge. Here, we chemically synthesized three homogeneously modified isoforms of Aβ (1-42) peptides bearing Tyr10 O-glycosylation, an unusual PTM initially identified from the cerebrospinal fluid samples of AD patients. We discovered that O-glycans significantly affect both the aggregation and degradation of Aβ42. By combining cryo-EM and various biochemical assays, we demonstrate that a Galβ1-3GalNAc modification redirects Aβ42 to form a new fibril polymorphic structure that is less stable and more vulnerable to Aβ-degrading enzymes (e.g., insulin-degrading enzyme). Thus, beyond showing how particular O-glycosylation modifications affect Aβ42 aggregation at the molecular level, our study provides powerful experimental tools to support further investigations about how PTMs affect Aβ42 fibril aggregation and AD-related neurotoxicity.

DOI: https://doi.org/10.1021/jacs.1c08607
Nat Commun. 2021 Dec 03. 12(1): 6941

Inhibition of cytoplasmic EZH2 induces antitumor activity through stabilization of the DLC1 tumor suppressor protein.

Brajendra K Tripathi, Meghan F Anderman, Disha Bhargava, Luciarita Boccuzzi, Xiaolan Qian, Dunrui Wang, Marian E Durkin, Alex G Papageorge, Fernando J de Miguel, Katerina Politi, Kylie J Walters, James H Doroshow, Douglas R Lowy.

mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.

DOI: https://doi.org/10.1038/s41467-021-26993-3
FEBS J. 2021 Dec 04.

A Guide to Studying Protein Aggregation.

Joëlle A J Housmans, Guiqin Wu, Joost Schymkowitz, Frederic Rousseau.

  Disrupted protein folding or decreased protein stability can lead to the accumulation of (partially) un- or misfolded proteins, which ultimately cause the formation of protein aggregates. Much of the interest in protein aggregation is associated with its involvement in a wide range of human diseases and the challenges it poses for large-scale biopharmaceutical manufacturing and formulation of therapeutic proteins and peptides. On the other hand, protein aggregates can also be functional, as observed in nature, which triggered its use in the development of biomaterials or therapeutics as well as for the improvement of food characteristics. Thus, unmasking the various steps involved in protein aggregation is critical to obtain a better understanding of the underlying mechanism of amyloid formation. This knowledge will allow a more tailored development of diagnostic methods and treatments for amyloid-associated diseases, as well as applications in the fields of new (bio)materials, food technology and therapeutics. However, the complex and dynamic nature of the aggregation process makes the study of protein aggregation challenging. To provide guidance on how to analyze protein aggregation, in this review we summarize the most commonly investigated aspects of protein aggregation with some popular corresponding methods.

Keywords:  Protein aggregation; aggregation kinetics; aggregation propensity; aggregation-prone region; amorphous aggregates; fibrils; protein homeostasis; protein stability; β-sheet

DOI:  https://doi.org/10.1111/febs.16312