TAK1-inhibitors did not reduce disease burden in a Vκ*MYC model of multiple myeloma.

OBJECTIVE: Multiple myeloma is a haematological malignancy characterized by proliferation of monoclonal plasma cells in the bone marrow. Development of resistance and minimal residual disease remain challenging in the treatment of multiple myeloma. Transforming growth factor-ß activated kinase 1 (TAK1) has recently gained attention as a potential drug target in multiple myeloma. This study aimed at determining the in vivo effects of TAK1-inhibitors in a Vκ*MYC multiple myeloma mouse model. RESULTS: We treated mice carrying Vκ*MYC multiple myeloma cells with the TAK1-inhibitors 5Z-7-oxozeaenol and NG25. There were tendencies towards increased survival for both inhibitors, but only NG25 prolonged survival significantly. However, this effect was limited, and no differences in disease burden were observed for any of the treatments. In conclusion, although TAK1-inhibitors might prolong survival somewhat, they do not prevent disease in the Vκ*MYC mouse model of multiple myeloma.

TAK1-inhibitors are cytotoxic for multiple myeloma cells alone and in combination with melphalan.

Multiple myeloma (MM) is an incurable cancer caused by malignant transformation of plasma cells. Transforming growth factor-ß activated kinase 1 (MAP3K7, TAK1) is a major regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. Both NF-κB and MAPK control expression of genes with vital roles for drug resistance in MM. TAK1 is an attractive drug target as it switches these survival pathways to cell death. Our analysis showed that patients with high MAP3K7 expression in the tumor had shorter overall and progression free survival. The TAK1-inhibitors NG25 and 5Z-7-oxozeaenol (5Z-7) were cytotoxic to MM cell lines and patient cells. NG25 reduced expression of MYC and E2F controlled genes, involved in tumor cell growth, cell cycle progression and drug resilience. TAK1 can be activated by genotoxic stress. NG25 and 5Z-7 induced both synergistic and additive cytotoxicity in combination with the alkylating agent melphalan. Melphalan activated TAK1, NF-κB, and the MAPKs p38 and c-Jun N-terminal kinase (JNK), as well as a transcriptional UV-response. This was blocked by NG25, and instead apoptosis was activated. MM induce elevated bone-degradation resulting in myeloma bone disease (MBD), which is the main cause of disability and morbidity in MM patients. NG25 and 5Z-7 reduced differentiation and viability of human bone degrading osteoclasts, suggesting that TAK1-inhibition can have a double beneficial effect for patients. In sum, TAK1 is a promising drug target for MM treatment.

Smac-mimetics reduce numbers and viability of human osteoclasts.

Elevated activity of bone-degrading osteoclasts (OC) contributes to pathological bone degradation in diseases such as multiple myeloma. Several proinflammatory cytokines, including TNF, contribute to osteoclastogenesis. The receptor-interacting protein kinase 1 (RIPK1) regulates inflammation and cell death. It is recruited to the TNF-receptor complex, where it is ubiquitinated, and activates transcription factor NF-κB and mitogen-activated protein kinases (MAPK). Smac-mimetics (SM) is a group of drugs that block RIPK1 ubiquitination and shifts RIPK1 to activation of apoptosis or necroptosis. In this manuscript, we show that the two SM birinapant and LCL-161 reduced the number and viability of primary human OC, and induced TNF-dependent cell death in OC precursors (pre-OC). Birinapant was more cytotoxic than LCL-161 and induced predominantly apoptosis and to some degree necroptosis. Both inhibitors restrained osteoclastogenesis induced by myeloma patient bone-marrow aspirates. SM has gained attention as novel treatment strategies both for cancer and chronic inflammatory pathologies, but limited information has been available on interactions with primary human immune cells. As LCL-161 is in phase 2 clinical studies for multiple myeloma, we propose that SM might possess additional benefits in reducing bone degradation in myeloma patients. Taken together, we show that SM reduces human osteoclastogenesis, and that these compounds may represent promising drug candidates for pathological bone degradation.

The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2.

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.

Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death.

Limited proteolysis of gasdermin D (GSDMD) generates an N-terminal pore-forming fragment that controls pyroptosis in macrophages. GSDMD is processed via inflammasome-activated caspase-1 or -11. It is currently unknown whether macrophage GSDMD can be processed by other mechanisms. Here, we describe an additional pathway controlling GSDMD processing. The inhibition of TAK1 or IκB kinase (IKK) by the Yersinia effector protein YopJ elicits RIPK1- and caspase-8-dependent cleavage of GSDMD, which subsequently results in cell death. GSDMD processing also contributes to the NLRP3 inflammasome-dependent release of interleukin-1ß (IL-1ß). Thus, caspase-8 acts as a regulator of GSDMD-driven cell death. Furthermore, this study establishes the importance of TAK1 and IKK activity in the control of GSDMD cleavage and cytotoxicity.

Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1 localization.

The organization of the Golgi apparatus (GA) is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The GA associated GTPase ADP ribosylation factor related protein 1 (ARFRP1) was previously shown to require N-terminal acetylation for membrane association and based on its N-terminal sequence, it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to-trans-Golgi network (TGN) traffic. We observed that ARFRP1 shifted from a predominantly cis-Golgi and TGN localization to localizing both Golgi and non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30 depletion induces Golgi scattering and induces aberrant ARFRP1 Golgi localization.

A Role for Human N-alpha Acetyltransferase 30 (Naa30) in Maintaining Mitochondrial Integrity.

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC Nt-acetylates a large variety of proteins and is essential for mitochondrial integrity and function.

Hydroxychloroquine potentiates carfilzomib toxicity towards myeloma cells.

Cells degrade proteins either by proteasomes that clinically are targeted by for example bortezomib or carfilzomib, or by formation of autophagosomes and lysosomal degradation that can be inhibited by hydroxychloroquine (HCQ). Multiple myeloma is unique among cancers because proteasomal inhibition has good clinical effects. However, some multiple myeloma patients display intrinsic resistance to the treatment and most patients acquire resistance over time. We hypothesized that simultaneous targeting both arms of protein degradation could be a way to improve treatment of multiple myeloma. Here we tested the combined effects of the lysosomal inhibitor HCQ and clinically relevant proteasome inhibitors on myeloma cell lines and primary cells. Carfilzomib and bortezomib both induced immunoglobulin-containing aggregates in myeloma cells. HCQ significantly potentiated the effect of carfilzomib in both cell lines and in primary myeloma cells. In contrast, HCQ had little or no effects on the toxicity of bortezomib. Furthermore, cells adapted to tolerate high levels of carfilzomib could be re-sensitized to the drug by co-treatment with HCQ. Thus, we show that inhibition of lysosomal degradation can overcome carfilzomib resistance, suggesting that the role of autophagy in myeloma cells is dependent on type of proteasome inhibitor. In conclusion, attempts should be made to combine HCQ with carfilzomib in the treatment of multiple myeloma.

Manipulation of Interleukin-1ß and Interleukin-18 Production by Yersinia pestis Effectors YopJ and YopM and Redundant Impact on Virulence.

Innate immunity plays a central role in resolving infections by pathogens. Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1ß and IL-18. These cytokines are produced by a two-step mechanism involving NF-κB-mediated pro-cytokine production and inflammasome-driven maturation into bioactive inflammatory mediators. Because of the anti-microbial effects induced by IL-1ß/IL-18, it may be desirable for pathogens to manipulate their production. Y. pestis type III secretion system effectors YopJ and YopM can interfere with different parts of this process. Both effectors have been reported to influence inflammasome caspase-1 activity; YopJ promotes caspase-8-dependent cell death and caspase-1 cleavage, whereas YopM inhibits caspase-1 activity via an incompletely understood mechanism. However, neither effector appears essential for full virulence in vivo Here we report that the sum of influences by YopJ and YopM on IL-1ß/IL-18 release is suppressive. In the absence of YopM, YopJ minimally affects caspase-1 cleavage but suppresses IL-1ß, IL-18, and other cytokines and chemokines. Importantly, we find that Y. pestis containing combined deletions of YopJ and YopM induces elevated levels of IL-1ß/IL-18 in vitro and in vivo and is significantly attenuated in a mouse model of bubonic plague. The reduced virulence of the YopJ-YopM mutant is dependent on the presence of IL-1ß, IL-18, and caspase-1. Thus, we conclude that Y. pestis YopJ and YopM can both exert a tight control of host IL-1ß/IL-18 production to benefit the bacteria, resulting in a redundant impact on virulence.

An organellar nα-acetyltransferase, naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.

N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

Protein N-terminal acetyltransferases: when the start matters.

The majority of eukaryotic proteins are subjected to N-terminal acetylation (Nt-acetylation), catalysed by N-terminal acetyltransferases (NATs). Recently, the structure of an NAT-peptide complex was determined, and detailed proteome-wide Nt-acetylation patterns were revealed. Furthermore, Nt-acetylation just emerged as a multifunctional regulator, acting as a protein degradation signal, an inhibitor of endoplasmic reticulum (ER) translocation, and a mediator of protein complex formation. Nt-acetylation is regulated by acetyl-coenzyme A (Ac-CoA) levels, and thereby links metabolic cell states to cell death. The essentiality of NATs in humans is stressed by the recent discovery of a human hereditary lethal disease caused by a mutation in an NAT gene. Here, we discuss how these recent findings shed light on NATs as major protein regulators and key cellular players.

The chaperone-like protein HYPK acts together with NatA in cotranslational N-terminal acetylation and prevention of Huntingtin aggregation.

The human NatA protein N(alpha)-terminal-acetyltransferase complex is responsible for cotranslational N-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry, we identified endogenous HYPK, a Huntingtin (Htt)-interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p, and hNaa15p were associated with polysome fractions, indicating a function of HYPK associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels, possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G(0)/G(1) phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-enhanced green fluorescent protein (Htt-EGFP) fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the data indicate that the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and for N-terminal acetylation.

Composition and biological significance of the human Nalpha-terminal acetyltransferases.

Protein Nalpha-terminal acetylation is one of the most common protein modifications in eukaryotic cells, occurring on approximately 80% of soluble human proteins. An increasing number of studies links Nalpha-terminal acetylation to cell differentiation, cell cycle, cell survival, and cancer. Thus, Nalpha-terminal acetylation is an essential modification for normal cell function in humans. Still, little is known about the functional role of Nalpha-terminal acetylation. Recently, the three major human N-acetyltransferase complexes, hNatA, hNatB and hNatC, were identified and characterized. We here summarize the identified N-terminal acetyltransferase complexes in humans, and we review the biological studies on Nalpha-terminal acetylation in humans and other higher eukaryotes.

A novel human NatA Nalpha-terminal acetyltransferase complex: hNaa16p-hNaa10p (hNat2-hArd1).

BACKGROUND: Protein acetylation is among the most common protein modifications. The two major types are post-translational Nepsilon-lysine acetylation catalyzed by KATs (Lysine acetyltransferases, previously named HATs (histone acetyltransferases) and co-translational Nalpha-terminal acetylation catalyzed by NATs (N-terminal acetyltransferases). The major NAT complex in yeast, NatA, is composed of the catalytic subunit Naa10p (N alpha acetyltransferase 10 protein) (Ard1p) and the auxiliary subunit Naa15p (Nat1p). The NatA complex potentially acetylates Ser-, Ala-, Thr-, Gly-, Val- and Cys- N-termini after Met-cleavage. In humans, the homologues hNaa15p (hNat1) and hNaa10p (hArd1) were demonstrated to form a stable ribosome associated NAT complex acetylating NatA type N-termini in vitro and in vivo. RESULTS: We here describe a novel human protein, hNaa16p (hNat2), with 70% sequence identity to hNaa15p (hNat1). The gene encoding hNaa16p originates from an early vertebrate duplication event from the common ancestor of hNAA15 and hNAA16. Immunoprecipitation coupled to mass spectrometry identified both endogenous hNaa15p and hNaa16p as distinct interaction partners of hNaa10p in HEK293 cells, thus demonstrating the presence of both hNaa15p-hNaa10p and hNaa16p-hNaa10p complexes. The hNaa16p-hNaa10p complex acetylates NatA type N-termini in vitro. hNaa16p is ribosome associated, supporting its potential role in cotranslational Nalpha-terminal acetylation. hNAA16 is expressed in a variety of human cell lines, but is generally less abundant as compared to hNAA15. Specific knockdown of hNAA16 induces cell death, suggesting an essential role for hNaa16p in human cells. CONCLUSION: At least two distinct NatA protein Nalpha-terminal acetyltransferases coexist in human cells potentially creating a more complex and flexible system for Nalpha-terminal acetylation as compared to lower eukaryotes.

Knockdown of human N alpha-terminal acetyltransferase complex C leads to p53-dependent apoptosis and aberrant human Arl8b localization.

Protein N(alpha)-terminal acetylation is one of the most common protein modifications in eukaryotic cells. In yeast, three major complexes, NatA, NatB, and NatC, catalyze nearly all N-terminal acetylation, acetylating specific subsets of protein N termini. In human cells, only the NatA and NatB complexes have been described. We here identify and characterize the human NatC (hNatC) complex, containing the catalytic subunit hMak3 and the auxiliary subunits hMak10 and hMak31. This complex associates with ribosomes, and hMak3 acetylates Met-Leu protein N termini in vitro, suggesting a model in which the human NatC complex functions in cotranslational N-terminal acetylation. Small interfering RNA-mediated knockdown of NatC subunits results in p53-dependent cell death and reduced growth of human cell lines. As a consequence of hMAK3 knockdown, p53 is stabilized and phosphorylated and there is a significant transcriptional activation of proapoptotic genes downstream of p53. Knockdown of hMAK3 alters the subcellular localization of the Arf-like GTPase hArl8b, supporting that hArl8b is a hMak3 substrate in vivo. Taken together, hNatC-mediated N-terminal acetylation is important for maintenance of protein function and cell viability in human cells.

Identification of the human N(alpha)-acetyltransferase complex B (hNatB): a complex important for cell-cycle progression.

Protein N(alpha)-terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, protein N(alpha)-terminal acetylation is performed by the N-acetyltransferase complexes NatA, NatB and NatC. In humans, only the NatA complex has been identified and characterized. In the present study we present the components of hNatB (human NatB complex). It consists of the Nat3p homologue hNAT3 (human N-acetyltransferase 3) and the Mdm20p homologue hMDM20 (human mitochondrial distribution and morphology 20). They form a stable complex and in vitro display sequence-specific N(alpha)-acetyltransferase activity on a peptide with the N-terminus Met-Asp-. hNAT3 and hMDM20 co-sediment with ribosomal pellets, thus supporting a model where hNatB acts co-translationally on nascent polypeptides. Specific knockdown of hNAT3 and hMDM20 disrupts normal cell-cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL-62. hNAT3 knockdown results in an increase in G(0)/G(1)-phase cells, whereas hMDM20 knockdown decreased the fraction of cells in G(0)/G(1)-phase and increased the fraction of cells in the sub-G(0)/G(1)-phase. In summary, we show for the first time a vertebrate NatB protein N(alpha)-acetyltransferase complex essential for normal cell proliferation.