Additive efficacy of a bispecific anti-TNF/IL-6 nanobody compound in translational models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases affecting primarily the joints. Despite successful therapies including antibodies against tumor necrosis factor (TNF) and interleukin-6 (IL-6) receptor, only 20 to 30% of patients experience remission. We studied whether inhibiting both TNF and IL-6 would result in improved efficacy. Using backtranslation from single-cell RNA sequencing (scRNA-seq) data from individuals with RA, we hypothesized that TNF and IL-6 act synergistically on fibroblast-like synoviocytes (FLS) and T cells. Coculture of FLS from individuals with RA and T cells supported this hypothesis, revealing effects on both disease-driving pathways and biomarkers. Combining anti-TNF and anti-IL-6 antibodies in collagen-induced arthritis (CIA) mouse models resulted in sustained long-term remission, improved histology, and effects on bone remodeling pathways. These promising data initiated the development of an anti-TNF/IL-6 bispecific nanobody compound 1, with similar potencies against TNF and IL-6. We observed additive efficacy of compound 1 in a FLS/T cell coculture affecting arthritis and T helper 17 (TH17) pathways. This nanobody compound transcript signature inversely overlapped with described RA endotypes, indicating a potential efficacy in a broader patient population. In summary, we showed superiority of a bispecific anti-TNF/IL-6 nanobody compound or combination treatment over monospecific treatments in both in vitro and in vivo models. We anticipate improved efficacy in upcoming clinical studies.

Inflammatory caspases: targets for novel therapies.

This review provides an overview of the biochemistry and activation of inflammatory caspases, and focuses on their therapeutic potential as disease targets in pathologies such as sepsis, Crohn's disease, rheumatoid arthritis, traumatic brain injury and amyotrophic lateral sclerosis (ALS). We summarize the proof-of-principal evidence obtained by studies in several corresponding experimental disease models confirming the validity of strategies targeting inflammatory caspases. We discuss the use of inflammatory caspase inhibitors, such as VX-740 (Pralnacasan) and VX-765, in clinical studies for rheumatoid arthritis and osteoarthritis. Finally, we point out recent approaches identifying novel peptidomimetic or non-peptide caspase inhibitors with suitable clinical profiles.

A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis.

The upstream of N-Ras (Unr) protein is involved in translational regulation of specific genes. For example, the Unr protein contributes to translation mediated by several viral and cellular internal ribosome entry sites (IRESs), including the PITSLRE IRES, which is activated at mitosis. Previously, we have shown that translation of the Unr mRNA itself can be initiated through an IRES. Here, we show that UNR mRNA translation and UNR IRES activity are significantly increased during mitosis. Functional analysis identified hnRNP C1/C2 proteins as UNR IRES stimulatory factors, whereas both polypyrimidine tract-binding protein (PTB) and Unr were found to function as inhibitors of UNR IRES-mediated translation. The increased UNR IRES activity during mitosis results from enhanced binding of the stimulatory hnRNP C1/C2 proteins and concomitant dissociation of PTB and Unr from the UNR IRES RNA. Our data suggest the existence of an IRES-dependent cascade in mitosis comprising hnRNP C1/C2 proteins that stimulate Unr expression, and Unr, in turn, contributes to PITSLRE IRES activity. The observation that RNA interference-mediated knockdown of hnRNP C1/C2 and Unr, respectively, abrogates and retards mitosis points out that regulation of IRES-mediated translation by hnRNP C1/C2 and Unr might be important in mitosis.

LIND/ABIN-3 is a novel lipopolysaccharide-inducible inhibitor of NF-kappaB activation.

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-kappaB (NF-kappaB). Although LPS-induced activation of NF-kappaB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-kappaB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-kappaB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-kappaB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-kappaB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKbeta. NF-kappaB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-kappaB activity in the liver, thereby partially protecting mice against LPS/D-(+)-galactosamine-induced mortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-kappaB activation.

Caspase-containing complexes in the regulation of cell death and inflammation.

Caspases are a family of cysteine proteases that are essential in the initiation and execution of apoptosis and the proteolytic maturation of inflammatory cytokines such as IL-1beta and IL-18. Caspases can be subdivided into those that have a large prodomain and those that have not. In general, apoptotic and inflammatory signalling pathways are initiated when large-prodomain caspases are recruited to large protein complexes via homotypic interactions involving death domain folds. The formation of these specialised multimeric platforms involves three major functions: (1) the sensing of cellular stress, damage, infection or inflammation; (2) multimerisation of the platform; and (3) recruitment and conformational activation of caspases. In this overview we discuss the complexes implicated in the regulation of cell death and inflammatory processes such as the death-inducing signalling complex (DISC), the apoptosome, the inflammasomes and the PIDDosome. We describe their sensing functions, compositions and functional outcomes. Inhibitory protein families such as FLIPs and CARD-only proteins prevent the recruitment of caspases in these sensing complexes, avoiding inappropriate initiation of cell death or inflammation.

UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein.

Upstream of N-ras (Unr) has been described as an internal initiation trans-acting factor (ITAF) in the cap-independent translation of some particular viral and cellular mRNAs. Two factors led us to hypothesize that the UNR 5'-untranslated region (5'-UTR) may contain an internal ribosome entry site (IRES). The first was the requirement for persisting Unr expression under conditions that correlate with cap-independent translation. The other was the observation that the primary UNR transcript contains a 447 nt long 5'-UTR including two upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Here we report that the UNR 5'-UTR allows IRES-dependent translation, as revealed by a dicistronic reporter assay. Various controls ruled out the contribution of leaky scanning, cryptic promoter sequences or RNA processing events to the ability of the UNR 5'-UTR to mediate internal initiation of translation. Ultraviolet cross-linking analysis and RNA affinity chromatography revealed the binding of polypyrimidine tract binding protein (PTB) to the UNR IRES, requiring a pyrimidine-rich region (nucleotides 335-355). Whereas overexpression of PTB in several cell lines inhibited UNR IRES activity and UNR protein expression, depletion of endogenous PTB using RNAi increased UNR IRES activity. Moreover, a mutant version of the UNR IRES lacking the PTB binding site was more efficient at directing IRES-mediated translation. In conclusion, our results demonstrate that translation of the ITAF Unr can itself be regulated by an IRES that is downregulated by PTB.

Apoptosis of hematopoietic cells induced by growth factor withdrawal is associated with caspase-9 mediated cleavage of Raf-1.

The Raf-1 serine/threonine kinase is a key protein that is implicated in the transmission of many growth and cell survival signals. In the present study we demonstrate that apoptosis of hematopoietic cells induced by IL-3-deprivation is associated with the cleavage of Raf-1, resulting in the separation of the N-terminal regulatory domain and the C-terminal kinase domain. Raf-1 cleavage specifically occurs upon triggering of the mitochondrial death pathway, and coincides with the activation of specific caspases. Moreover, Bcl-2 overexpression or treatment with the caspase inhibitor z-VAD.fmk completely prevented Raf-1 cleavage, whereas caspase inhibition by treatment of cells with Ac-DEVD.fmk or z-IETD.fmk, or CrmA overexpression had no effect. Furthermore, in vitro cleavage studies indicate that caspase-9, which is the apical protease in the mitochondrial death pathway, is able to cleave Raf-1 at position D279. Cell fractionation studies showed that the Raf-1 C-terminal fragment that is generated upon IL-3 withdrawal is localized predominantly to the mitochondria. In addition, constitutive expression of this C-terminal Raf-1 fragment fused to a mitochondrial targeting sequence in Ba/F3 pre-B cells significantly delays apoptosis induced by IL-3 withdrawal. These results suggest an important role for caspase-9 mediated cleavage of Raf-1 in the negative feedback regulation of hematopoietic cell apoptosis induced by growth factor withdrawal.

The polypyrimidine tract-binding protein stimulates HIF-1alpha IRES-mediated translation during hypoxia.

When oxygen supply is restricted, protein synthesis is rapidly abrogated owing to inhibition of global translation. However, HIF-1alpha protein expression can persist during hypoxia, owing to an internal ribosome entry site (IRES) in the 5'-untranslated region of its mRNA. Here, we report on the molecular mechanism of HIF-1alpha IRES-mediated translation during oxygen deprivation. Using RNA affinity chromatography and UV-crosslinking experiments, we show that the polypyrimidine tract binding protein (PTB) can specifically interact with the HIF-1alpha IRES, and that this interaction is enhanced in hypoxic conditions. Overexpression of PTB enhanced HIF-1alpha IRES activity, whereas RNA interference-mediated downregula-tion of PTB protein expression inhibited HIF-1alpha IRES activity. Furthermore, hypoxia-induced stimulation of the HIF-1alpha IRES was reduced in cells in which PTB function was downregulated. In agreement with these results, the IRES activity of HIF-1alpha IRES deletion mutants that are deficient in PTB-binding could not be stimulated by oxygen deprivation. All together, our data suggest that PTB plays a stimulatory role in the IRES-mediated translation of HIF-1alpha when oxygen supply is limited.

Regulation of the cell-cycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2alpha.

The PITSLRE kinases belong to the large family of cyclin-dependent protein kinases. Their function has been related to cell-cycle regulation, splicing and apoptosis. We have previously shown that the open reading frame of the p110(PITSLRE) transcript contains an IRES (internal ribosome entry site) that allows the expression of a smaller p58(PITSLRE) isoform during the G2/M stage of the cell cycle. In the present study we investigated further the role of cis- and trans-acting factors in the regulation of the PITSLRE IRES. Progressive deletion analysis showed that both a purine-rich sequence and a Unr (upstream of N-ras) consensus binding site are essential for PITSLRE IRES activity. In line with these observations, we demonstrate that the PITSLRE IRES interacts with the Unr protein, which is more prominently expressed at the G2/M stage of the cell cycle. We also show that phosphorylation of the alpha-subunit of the canonical initiation factor eIF-2 is increased at G2/M. Interestingly, phosphorylation of eIF-2alpha has a permissive effect on the efficiency of both the PITSLRE IRES and the ornithine decarboxylase IRES, two cell cycle-dependent IRESs, in mediating internal initiation of translation, whereas this was not observed with the viral EMCV (encephalomyocarditis virus) and HRV (human rhinovirus) IRESs.