Signal peptide peptidase-like 2b modulates the amyloidogenic pathway and exhibits an Aß-dependent expression in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disorder driven by abnormal amyloid ß-peptide (Aß) levels. In this study, we investigated the role of presenilin-like signal peptide peptidase-like 2b (SPPL2b) in AD pathophysiology and its potential as a druggable target within the Aß cascade. Exogenous Aß42 influenced SPPL2b expression in human cell lines and acute mouse brain slices. SPPL2b and its AD-related substrate BRI2 were evaluated in the brains of AppNL-G-F knock-in AD mice and human postmortem AD brains. An early high cortical expression of SPPL2b was observed, followed by a downregulation in late AD pathology in AppNL-G-F mice, correlating with synaptic loss. To understand the consequences of pathophysiological SPPL2b dysregulation, we found that SPPL2b overexpression significantly increased APP cleavage, while genetic deletion reduced APP cleavage and Aß production. Notably, postmortem AD brains showed higher levels of SPPL2b's BRI2 substrate compared to healthy control samples. These results strongly support the involvement of SPPL2b in AD pathology. The early Aß-induced upregulation of SPPL2b may enhance Aß production in a vicious cycle, further aggravating Aß pathology. Therefore, SPPL2b emerges as a potential anti-Aß drug target.

The role of SPP/SPPL intramembrane proteases in membrane protein homeostasis.

Signal peptide peptidase (SPP) and the four SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 constitute a family of aspartyl intramembrane proteases with homology to presenilins. The different members reside in distinct cellular localisations within the secretory pathway and the endo-lysosomal system. Despite individual cleavage characteristics, they all cleave single-span transmembrane proteins with a type II orientation exhibiting a cytosolic N-terminus. Though the identification of substrates is not complete, SPP/SPPL-mediated proteolysis appears to be rather selective. Therefore, according to our current understanding cleavage by SPP/SPPL proteases rather seems to serve a regulatory function than being a bulk proteolytic pathway. In the present review, we will summarise our state of knowledge on SPP/SPPL proteases and in particular highlight recently identified substrates and the functional and/or (patho)-physiological implications of these cleavage events. Based on this, we aim to provide an overview of the current open questions in the field. These are connected to the regulation of these proteases at the cellular level but also in context of disease and patho-physiological processes. Furthermore, the interplay with other proteostatic systems capable of degrading membrane proteins is beginning to emerge.

Accelerating development of engineered T cell therapies in the EU: current regulatory framework for studying multiple product versions and T2EVOLVE recommendations.

To accelerate the development of Advanced Therapy Medicinal Products (ATMPs) for patients suffering from life-threatening cancer with limited therapeutic options, regulatory approaches need to be constantly reviewed, evaluated and adjusted, as necessary. This includes utilizing science and risk-based approaches to mitigate and balance potential risks associated with early clinical research and a more flexible manufacturing paradigm. In this paper, T2EVOLVE an Innovative Medicine Initiative (IMI) consortium explores opportunities to expedite the development of CAR and TCR engineered T cell therapies in the EU by leveraging tools within the existing EU regulatory framework to facilitate an iterative and adaptive learning approach across different product versions with similar design elements or based on the same platform technology. As understanding of the linkage between product quality attributes, manufacturing processes, clinical efficacy and safety evolves through development and post licensure, opportunities are emerging to streamline regulatory submissions, optimize clinical studies and extrapolate data across product versions reducing the need to perform duplicative studies. It is worth noting that this paper is focusing on CAR- and TCR-engineered T cell therapies but the concepts may be applied more broadly to engineered cell therapy products (e.g., CAR NK cell therapy products).

Genome-wide detection of human intronic AG-gain variants located between splicing branchpoints and canonical splice acceptor sites.

Human genetic variants that introduce an AG into the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing. Using our genome-wide BP database, we delineated the BP-ACC segments of all human introns and found extreme depletion of AG/YAG in the [BP+8, ACC-4] high-risk region. We developed AGAIN as a genome-wide computational approach to systematically and precisely pinpoint intronic AG-gain variants within the BP-ACC regions. AGAIN identified 350 AG-gain variants from the Human Gene Mutation Database, all of which alter splicing and cause disease. Among them, 74% created new acceptor sites, whereas 31% resulted in complete exon skipping. AGAIN also predicts the protein-level products resulting from these two consequences. We performed AGAIN on our exome/genomes database of patients with severe infectious diseases but without known genetic etiology and identified a private homozygous intronic AG-gain variant in the antimycobacterial gene SPPL2A in a patient with mycobacterial disease. AGAIN also predicts a retention of six intronic nucleotides that encode an in-frame stop codon, turning AG-gain into stop-gain. This allele was then confirmed experimentally to lead to loss of function by disrupting splicing. We further showed that AG-gain variants inside the high-risk region led to misspliced products, while those outside the region did not, by two case studies in genes STAT1 and IRF7. We finally evaluated AGAIN on our 14 paired exome-RNAseq samples and found that 82% of AG-gain variants in high-risk regions showed evidence of missplicing. AGAIN is publicly available from https://hgidsoft.rockefeller.edu/AGAIN and https://github.com/casanova-lab/AGAIN.

Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling.

More than 20 years ago, signal peptide peptidase (SPP) and its homologues, the signal peptide peptidase-like (SPPL) proteases have been identified based on their sequence similarity to presenilins, a related family of intramembrane aspartyl proteases. Other than those for the presenilins, no high-resolution structures for the SPP/SPPL proteases are available. Despite this limitation, over the years bioinformatical and biochemical data have accumulated, which altogether have provided a picture of the overall structure and topology of these proteases, their localization in the cell, the process of substrate recognition, their cleavage mechanism, and their function. Recently, the artificial intelligence-based structure prediction tool AlphaFold has added high-confidence models of the expected fold of SPP/SPPL proteases. In this review, we summarize known structural aspects of the SPP/SPPL family as well as their substrates. Of particular interest are the emerging substrate recognition and catalytic mechanisms that might lead to the prediction and identification of more potential substrates and deeper insight into physiological and pathophysiological roles of proteolysis.

Dynamic association of the intramembrane proteases SPPL2a/b and their substrates with tetraspanin-enriched microdomains.

Signal peptide peptidase-like 2a and b (SPPL2a/b) are aspartyl intramembrane proteases and cleave tail-anchored proteins as well as N-terminal fragments (NTFs) derived from type II-oriented transmembrane proteins. How these proteases recruit substrates and cleavage is regulated, is still incompletely understood. We found that SPPL2a/b localize to detergent-resistant membrane (DRM) domains with the characteristics of tetraspanin-enriched microdomains (TEMs). Based on this, association with several tetraspanins was evaluated. We demonstrate that not only SPPL2a/b but also their substrates tumor necrosis factor (TNF) and CD74 associate with tetraspanins like CD9, CD81, and CD82 and/or TEMs and analyze the stability of these complexes in different detergents. CD9 and CD81 deficiency has protease- and substrate-selective effects on SPPL2a/b function. Our findings suggest that reciprocal interactions with tetraspanins may assist protease-substrate encounters of SPPL2a/b within the membrane. Beyond SPP/SPPL proteases, this supports previous concepts that tetraspanins facilitate membrane-embedded proteolytic processes.

The intramembrane proteases SPPL2a and SPPL2b regulate the homeostasis of selected SNARE proteins.

Signal peptide peptidase (SPP) and SPP-like (SPPL) aspartyl intramembrane proteases are known to contribute to sequential processing of type II-oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER-resident family members SPP and SPPL2c were shown to also cleave tail-anchored proteins, including selected SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co-expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail-anchored proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1-4 levels in tissues and primary cells of SPPL2a/b double-deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1-4 in a cell type- and tissue-dependent manner, identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1-4 by initiating the degradation of these proteins, which might impact cellular trafficking.

C11orf94/Frey is a key regulator for male fertility by controlling Izumo1 complex assembly.

Although gamete fusion represents the central event in sexual reproduction, the required protein machinery is poorly defined. In sperm cells, Izumo1 and several Izumo1-associated proteins play an essential role for this process. However, so far, the mechanisms underlying transport and maturation of Izumo1 and its incorporation into high molecular weight complexes are incompletely defined. Here, we provide a detailed characterization of the C11orf94 protein, which we rename Frey, which provides a platform for the assembly of Izumo1 complexes. By retaining Izumo1 in the endoplasmic reticulum, Frey facilitates its incorporation into high molecular weight complexes. To fulfill its function, the unstable Frey protein is stabilized within the catalytic center of an intramembrane protease. Loss of Frey results in reduced assembly of Izumo1 complexes and male infertility due to impaired gamete fusion. Collectively, these findings provide mechanistic insights into the early biogenesis and functional relevance of Izumo1 complexes.

Phagosomal signalling of the C-type lectin receptor Dectin-1 is terminated by intramembrane proteolysis.

Sensing of pathogens by pattern recognition receptors (PRR) is critical to initiate protective host defence reactions. However, activation of the immune system has to be carefully titrated to avoid tissue damage necessitating mechanisms to control and terminate PRR signalling. Dectin-1 is a PRR for fungal ß-glucans on immune cells that is rapidly internalised after ligand-binding. Here, we demonstrate that pathogen recognition by the Dectin-1a isoform results in the formation of a stable receptor fragment devoid of the ligand binding domain. This fragment persists in phagosomal membranes and contributes to signal transduction which is terminated by the intramembrane proteases Signal Peptide Peptidase-like (SPPL) 2a and 2b. Consequently, immune cells lacking SPPL2b demonstrate increased anti-fungal ROS production, killing capacity and cytokine responses. The identified mechanism allows to uncouple the PRR signalling response from delivery of the pathogen to degradative compartments and identifies intramembrane proteases as part of a regulatory circuit to control anti-fungal immune responses.

Signal peptide peptidase-like 2 proteases: Regulatory switches or proteasome of the membrane?

Signal peptide peptidase-like 2 (SPPL) proteases constitute a subfamily of SPP/SPPL intramembrane proteases which are homologues of the presenilins, the catalytic core of the γ-secretase complex. The three SPPL2 proteases SPPL2a, SPPL2b and SPPL2c proteolyse single-span, type II-oriented transmembrane proteins and/or tail-anchored proteins within their hydrophobic transmembrane segments. We review recent progress in defining substrate spectra and in vivo functions of these proteases. Characterisation of the respective knockout mice has implicated SPPL2 proteases in immune cell differentiation and function, prevention of atherosclerotic plaque development and spermatogenesis. Mechanisms how substrates are selected by these enzymes are still incompletely understood. We will discuss current views on how selective SPPL2-mediated cleavage is or whether these proteases may exhibit a generalised role in the turnover of membrane proteins. This has been suggested previously for the mechanistically related γ-secretase for which the term "proteasome of the membrane" has been coined based on its broad substrate spectrum. With regard to individual substrates, potential signalling functions of the resulting cytosolic cleavage fragments remain a controversial aspect. However, it has been clearly shown that SPPL2 proteases can influence cellular signalling and membrane trafficking by controlling levels of their membrane-bound substrate proteins which highlights these enzymes as regulatory switches. Based on this, regulatory mechanisms controlling activity of SPPL2 proteases would need to be postulated, which are just beginning to emerge. These different questions, which are relevant for other families of intramembrane proteases in a similar way, will be critically discussed based on the current state of knowledge.

Acclimation of intestinal morphology and function in Djungarian hamsters (Phodopus sungorus) related to seasonal and acute energy balance.

Small mammals exhibit seasonal changes in intestinal morphology and function via increased intestine size and resorptive surface and/or nutrient transport capacity to increase energy yield from food during winter. This study investigated whether seasonal or acute acclimation to anticipated or actual energetic challenges in Djungarian hamsters also resulted in higher nutrient resorption capacities owing to changes in small intestine histology and physiology. The hamsters show numerous seasonal energy-saving adjustments in response to short photoperiod. As spontaneous daily torpor represents one of these adjustments related to food quality and quantity, it was hypothesized that the hamsters' variable torpor expression patterns are influenced by their individual nutrient uptake capacity. Hamsters under short photoperiod showed longer small intestines and higher mucosal electrogenic transport capacities for glucose relative to body mass. Similar observations were made in hamsters under long photoperiod and food restriction. However, this acute energetic challenge caused a stronger increase of glucose transport capacity. Apart from that, neither fasting-induced torpor in food-restricted hamsters nor spontaneous daily torpor in short photoperiod-exposed hamsters clearly correlated with mucosal glucose transport capacity. Both seasonally anticipated and acute energetic challenges caused adjustments in the hamsters' small intestine. Short photoperiod appeared to induce an integration of these and other acclimation processes in relation to body mass to achieve a long-term adjustment of energy balance. Food restriction seemed to result in a more flexible, short-term strategy of maximizing energy uptake possibly via mucosal glucose transport and reducing energy consumption via torpor expression as an emergency response.

Deficiency of the Intramembrane Protease SPPL2a Alters Antimycobacterial Cytokine Responses of Dendritic Cells.

Signal peptide peptidase-like 2a (SPPL2a) is an aspartyl intramembrane protease essential for degradation of the invariant chain CD74. In humans, absence of SPPL2a leads to Mendelian susceptibility to mycobacterial disease, which is attributed to a loss of the dendritic cell (DC) subset conventional DC2. In this study, we confirm depletion of conventional DC2 in lymphatic tissues of SPPL2a-/- mice and demonstrate dependence on CD74 using SPPL2a-/- CD74-/- mice. Upon contact with mycobacteria, SPPL2a-/- bone marrow-derived DCs show enhanced secretion of IL-1ß, whereas production of IL-10 and IFN-ß is reduced. These effects correlated with modulated responses upon selective stimulation of the pattern recognition receptors TLR4 and Dectin-1. In SPPL2a-/- bone marrow-derived DCs, Dectin-1 is redistributed to endosomal compartments. Thus, SPPL2a deficiency alters pattern recognition receptor pathways in a CD74-dependent way, shifting the balance from anti- to proinflammatory cytokines in antimycobacterial responses. We propose that in addition to the DC reduction, this altered DC functionality contributes to Mendelian susceptibility to mycobacterial disease upon SPPL2a deficiency.

Cathepsin S provokes interleukin-6 (IL-6) trans-signaling through cleavage of the IL-6 receptor in vitro.

The cytokine interleukin-6 (IL-6) fulfills its pleiotropic functions via different modes of signaling. Regenerative and anti-inflammatory activities are mediated via classic signaling, in which IL-6 binds to the membrane-bound IL-6 receptor (IL-6R). For IL-6 trans-signaling, which accounts for the pro-inflammatory properties of the cytokine, IL-6 activates its target cells via soluble forms of the IL-6R (sIL-6R). We have previously shown that the majority of sIL-6R in human serum originates from proteolytic cleavage and mapped the cleavage site of the IL-6R. The cleavage occurs between Pro-355 and Val-356, which is the same cleavage site that the metalloprotease ADAM17 uses in vitro. However, sIL-6R serum levels are unchanged in hypomorphic ADAM17ex/ex mice, making the involvement of ADAM17 questionable. In order to identify other proteases that could be relevant for sIL-6R generation in vivo, we perform a screening approach based on the known cleavage site. We identify several candidate proteases and characterize the cysteine protease cathepsin S (CTSS) in detail. We show that CTSS is able to cleave the IL-6R in vitro and that the released sIL-6R is biologically active and can induce IL-6 trans-signaling. However, CTSS does not use the Pro-355/Val-356 cleavage site, and sIL-6R serum levels are not altered in Ctss-/- mice. In conclusion, we identify a novel protease of the IL-6R that can induce IL-6 trans-signaling, but does not contribute to steady-state sIL-6R serum levels.

Physiological functions of SPP/SPPL intramembrane proteases.

Intramembrane proteolysis describes the cleavage of substrate proteins within their hydrophobic transmembrane segments. Several families of intramembrane proteases have been identified including the aspartyl proteases Signal peptide peptidase (SPP) and its homologues, the SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3. As presenilin homologues, they employ a similar catalytic mechanism as the well-studied γ-secretase. However, SPP/SPPL proteases cleave transmembrane proteins with a type II topology. The characterisation of SPP/SPPL-deficient mouse models has highlighted a still growing spectrum of biological functions and also promoted the substrate discovery of these proteases. In this review, we will summarise the current hypotheses how phenotypes of these mouse models are linked to the molecular function of the enzymes. At the cellular level, SPP/SPPL-mediated cleavage events rather provide specific regulatory switches than unspecific bulk proteolysis. By this means, a plethora of different cell biological pathways is influenced including signal transduction, membrane trafficking and protein glycosylation.

Proteolytic Regulation of the Lectin-Like Oxidized Lipoprotein Receptor LOX-1.

The lectin-like oxidized-LDL (oxLDL) receptor LOX-1, which is broadly expressed in vascular cells, represents a key mediator of endothelial activation and dysfunction in atherosclerotic plaque development. Being a member of the C-type lectin receptor family, LOX-1 can bind different ligands, with oxLDL being the best characterized. LOX-1 mediates oxLDL uptake into vascular cells and by this means can promote foam cell formation. In addition, LOX-1 triggers multiple signaling pathways, which ultimately induce a pro-atherogenic and pro-fibrotic transcriptional program. However, the molecular mechanisms underlying this signal transduction remain incompletely understood. In this regard, proteolysis has recently emerged as a regulatory mechanism of LOX-1 function. Different proteolytic cleavages within the LOX-1 protein can initiate its turnover and control the cellular levels of this receptor. Thereby, cleavage products with individual biological functions and/or medical significance are produced. Ectodomain shedding leads to the release of a soluble form of the receptor (sLOX1) which has been suggested to have diagnostic potential as a biomarker. Removal of the ectodomain leaves behind a membrane-bound N-terminal fragment (NTF), which despite being devoid of the ligand-binding domain is actively involved in signal transduction. Degradation of this LOX-1 NTF, which represents an athero-protective mechanism, critically depends on the aspartyl intramembrane proteases Signal peptide peptidase-like 2a and b (SPPL2a/b). Here, we present an overview of the biology of LOX-1 focusing on how proteolytic cleavages directly modulate the function of this receptor and, what kind of pathophysiological implications this has in cardiovascular disease.

Binary Mixtures of Ionic Liquids in Aqueous Solution: Towards an Understanding of their Salting-In/Salting-Out Phenomena.

The order of the salting-in or salting-out inducing ability of ions on the aqueous solubility of macromolecules in aqueous solutions is known as the Hofmeister series. Taking into account that ionic liquids (ILs) are constituted by ions, they can exert similar effects on the solubility of other ILs in aqueous media. In order to expand the knowledge on the salting-in/-out ability of ILs, experimental studies on the solubility of 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonylimide) in water in presence of other IL/salts were conducted at 298.15 K at atmospheric pressure. Both the impact of the anion and cation of the IL were evaluated with the following ILs/salts: 1-butyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium hydrogensulfate, cholinium bis(trifluoromethylsulfonyl)imide, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide and lithium bis(trifluoromethylsulfonyl)imide, in a wide composition range. As happens with common salts, both salting-in and salting-out effects exerted by ILs were observed, with a higher impact exerted by the IL anion on the salting-out phenomenon. These data allow to better understand the ILs impact when designing liquid-liquid separation processes.

Gut microbial transformation of the dietary mutagen MeIQx may reduce exposure levels without altering intestinal transport.

The mutagen and probable human carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is metabolized in the colon to 9-hydroxyl-2,7-dimethyl-7,9,10,11-tetrahydropyrimido[2',1':2,3]imidazo[4,5-f]quinoxaline (MeIQx-M1) by conjugation with microbially generated acrolein. However, whether this microbiota-controlled process alters systemic exposure and hepatotoxicity of MeIQx remains unclear. The physiological relevance of this microbial transformation on the systemic exposure of MeIQx was investigated using an in vitro-in vivo extrapolation approach. To address whether microbial transformation influences intestinal transport of MeIQx, the intestinal uptake of MeIQx and its metabolite MeIQx-M1 was quantified using Ussing chambers mounted with different intestinal segments from male Fischer 344 rats. Up to 0.4% of both MeIQx and MeIQx-M1 were transported from the mucosal side to the serosal side of intestinal tissue within 90 min, suggesting that the intestinal uptake of both compounds is similar. With the uptake rates of both compounds, physiologically based pharmacokinetic (PBPK) modeling of the fate of MeIQx in the human body including microbial transformation of MeIQx was performed. Results indicate for the first time that high levels of microbe-derived acrolein would be required to significantly reduce systemic exposure of MeIQx in humans. Finally, neither MeIQx nor MeIQx-M1 were cytotoxic towards human liver HepaRG cells at dietary or higher concentrations of MeIQx. In summary, these findings suggest that gut microbial transformation of heterocyclic amines has the potential to influence systemic human exposure to some extent, but may require significant gut microbial production of acrolein and that further investigations are needed to understand physiological levels of acrolein and competing biotransformation pathways.

Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis.

The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1-mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase-like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.