Inactive Variant Research Articles

Editorial: Bacterial Exotoxins: How Bacteria Fight the Immune System

Editorial: Bacterial Exotoxins: How Bacteria Fight the Immune System

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1β in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1β secretion. The apparent paradox of reduced IL-1β secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1β and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1β-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.

CORM-2 inhibits TXNIP/NLRP3 inflammasome pathway in LPS-induced acute lung injury.

Accumulated studies suggest that exogenously administered carbon monoxide is beneficial for the resolution of acute lung inflammation. The present study aimed to examine the effects and the underlying mechanisms of CORM-2 on thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome pathway in lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was intratracheally induced by LPS in C57BL6 mice. CORM-2 or iCORM-2 (30mg/kg i.p.) was administered immediately before LPS instillation. 6 h later, lung bronchoalveolar lavage (BAL) fluids were acquired for IL-18, IL-1β, and cell measurement, and lung issues were collected for histologic examination, wet/dry weight ratio, and determination of TXNIP/NLRP3 inflammasome expression, NLRP3 inflammasome and NF-ΚB activity, and reactive oxygen species (ROS) production. LPS triggered significant lung edema, lung injury, and leukocyte infiltration, and elevated the levels of IL-1β and IL-18 in lung BAL fluids. CORM-2 pretreatment resulted in a marked amelioration of lung injury and reduced IL-1β and IL-18 secretion in BAL fluids. In lung tissues; CORM-2 down-regulated mRNA and protein level of TXNIP, NLRP3, ASC, and caspase-1. Furthermore, CORM-2 reduced ROS production, inhibited NLRP3 inflammasome and NF-κB activity, and interaction of TXNIP-NLRP3. However, no significant differences were detected between the LPS and iCORM-2 (an inactive variant of CORM-2) group. CORM-2 suppresses TXNIP/NLRP3 inflammasome pathway and protects against LPS-induced lung injury.

Mechanism of endonuclease cleavage by the HigB toxin.

Bacteria encode multiple type II toxin–antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro. Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition.

Chronic Contractile Dysfunction without Hypertrophy Does Not Provoke a Compensatory Transcriptional Response in Mouse Hearts.

Diseased myocardium from humans and experimental animal models shows heightened expression and activity of a specific subtype of phospholipase C (PLC), the splice variant PLCβ1b. Previous studies from our group showed that increasing PLCβ1b expression in adult mouse hearts by viral transduction was sufficient to cause sustained contractile dysfunction of rapid onset, which was maintained indefinitely in the absence of other pathological changes in the myocardium. We hypothesized that impaired contractility alone would be sufficient to induce a compensatory transcriptional response. Unbiased, comprehensive mRNA-sequencing was performed on 6 biological replicates of rAAV6-treated blank, PLCβ1b and PLCβ1a (closely related but inactive splice variant) hearts 8 weeks after injection, when reduced contractility was manifest in PLCβ1b hearts without evidence of induced hypertrophy. Expression of PLCβ1b resulted in expression changes in only 9 genes at FDR<0.1 when compared with control and these genes appeared unrelated to contractility. Importantly, PLCβ1a caused similar mild expression changes to PLCβ1b, despite a complete lack of effect of this isoform on cardiac contractility. We conclude that contractile depression caused by PLCβ1b activation is largely independent of changes in the transcriptome, and thus that lowered contractility is not sufficient in itself to provoke measurable transcriptomic alterations. In addition, our data stress the importance of a stringent control group to filter out transcriptional changes unrelated to cardiac function.

Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter

The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous expression systems/model organisms for large and complex proteins are at a premium. We have verified the applicability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial subcellular localization and multiple proposed cellular functions. We were able to demonstrate its efficient expression in the expected subcellular locations as well as biochemical activity of the overexpressed protein (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the catalytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expression system to study the activity of membrane transporters from the ABC superfamily.

ADP-Ribose Activates the TRPM2 Channel from the Sea Anemone Nematostella vectensis Independently of the NUDT9H Domain.

The human redox-sensitive Transient receptor potential melastatin type 2 (hTRPM2) channel contains the C-terminal Nudix hydrolase domain NUDT9H which most likely binds ADP-ribose. During oxidative stress, the intracellular release of ADP-ribose triggers the activation of hTRPM2. The TRPM2 orthologue from Nematostella vectensis (nv) is also stimulated by ADP-ribose but not by the oxidant hydrogen peroxide. For further clarification of the structure-function relationships of these two distantly related channel orthologues, we performed whole-cell as well as single channel patch-clamp recordings, Ca2+-imaging and Western blot analysis after heterologous expression of wild-type and mutated channels in HEK-293 cells. We demonstrate that the removal of the entire NUDT9H domain does not disturb the response of nvTRPM2 to ADP-ribose. The deletion, however, created channels that were activated by hydrogen peroxide, as did mutations within the NUDT9H domain of nvTRPM2 that presumably suppress its enzymatic function. The same findings were obtained with the nvTRPM2 channel when the NUDT9H domain was replaced by the corresponding sequences of the original hNUDT9 enzyme. Whenever the enzyme domain was mutated to presumably inactive variants, channel activation by hydrogen peroxide could be achieved. Moreover, we found strong evidences for ADPRase activity of the isolated NUDT9H domain of nvTRPM2 in co-expression experiments with the C-terminally truncated nvTRPM2 channel. Thus, there is a clear correlation between the loss of enzymatic activity and the capability of nvTRPM2 to respond to oxidative stress. In striking contrast, the channel function of the hTRPM2 orthologue, in particular its sensitivity to ADP-ribose, was abrogated by already small changes of the NUDT9H domain. These findings establish nvTRPM2 as a channel gated by ADP-ribose through a novel mechanism. We conclude that the endogenous NUDT9H domain does not directly affect ADP-ribose-dependent gating of the nvTRPM2 channel; instead it exerts an independent catalytic function which possibly controls the intracellular availability of ADP-ribose.

Splicing variants of ADAR2 and ADAR2-mediated RNA editing in glioma.

The roles of alternative splicing and RNA editing in gene regulation and transcriptome diversity are well documented. Adenosine deaminases acting on RNA (ADARs) are responsible for adenosine-to-inosine (A-to-I) editing and exemplify the complex association between RNA editing and alternative splicing. The self-editing activity of ADAR2, which acts on its own pre-mRNA, leads to its alternative splicing. Alternative splicing occurs independently at nine splicing sites on ADAR2 pre-mRNA, generating numerous alternative splicing variants with various catalytic activities. A-to-I RNA editing is important in a range of physiological processes in humans and is associated with several diseases, including amyotrophic lateral sclerosis, mood disorders, epilepsy and glioma. Reduced editing at the glutamine/arginine site of the AMPA receptor subunit GluA2 in glioma, without any alteration in ADAR2 expression, is a notable phenomenon. Several studies have tried to explain this alteration in the catalytic activity of ADAR2; however, the underlying mechanism remains unclear. The present review summarizes the relevant literature and shares experimental results concerning ADAR2 alternative splicing. In particular, the present review demonstrates that shifts in the relative abundance of the active and inactive splicing variants of ADAR2 may reduce the ADAR2 editing activity in glioma. Dominant expression of ADAR2 splicing variant with low enzyme activity causes reduced RNA editing of GluA2 subunit at the glutamine/arginine site in glioma.

Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses

One of the β-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-β-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-β-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 106 M−1. The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-β-glucanase.

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris.

The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal "glyco-stress" caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases.

Bacterial AvrRpt2-Like Cysteine Proteases Block Activation of the Arabidopsis Mitogen-Activated Protein Kinases, MPK4 and MPK11.

To establish infection, pathogens deliver effectors into host cells to target immune signaling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defense regulator, RPM1-INTERACTING PROTEIN4 (RIN4), and interference with plant auxin signaling. We show now that AvrRpt2 specifically suppresses the flagellin-induced phosphorylation of Arabidopsis (Arabidopsis thaliana) MPK4 and MPK11 but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defense genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called nitrate-induced family as well as from auxin signaling. Thus, this selective suppression of specific mitogen-activated protein kinases is independent of the previously known AvrRpt2 targets and potentially represents a novel virulence function of AvrRpt2.

The endoplasmic reticulum-mitochondria interface is perturbed in PARK2 knockout mice and patients with PARK2 mutations.

Mutations in PARK2, encoding the E3 ubiquitin protein ligase Parkin, are a common cause of autosomal recessive Parkinson's disease (PD). Loss of PARK2 function compromises mitochondrial quality by affecting mitochondrial biogenesis, bioenergetics, dynamics, transport and turnover. We investigated the impact of PARK2 dysfunction on the endoplasmic reticulum (ER)-mitochondria interface, which mediates calcium (Ca2+) exchange between the two compartments and is essential for Parkin-dependent mitophagy. Confocal and electron microscopy analyses showed the ER and mitochondria to be in closer proximity in primary fibroblasts from PARK2 knockout (KO) mice and PD patients with PARK2 mutations than in controls. Ca2+ flux to the cytosol was also modified, due to enhanced ER-to-mitochondria Ca2+ transfers, a change that was also observed in neurons derived from induced pluripotent stem cells of a patient with PARK2 mutations. Subcellular fractionation showed the abundance of the Parkin substrate mitofusin 2 (Mfn2), which is known to modulate the ER-mitochondria interface, to be specifically higher in the mitochondrion-associated ER membrane compartment in PARK2 KO tissue. Mfn2 downregulation or the exogenous expression of normal Parkin restored cytosolic Ca2+ transients in fibroblasts from patients with PARK2 mutations. In contrast, a catalytically inactive PD-related Parkin variant had no effect. Overall, our data suggest that Parkin is directly involved in regulating ER-mitochondria contacts and provide new insight into the role of the loss of Parkin function in PD development.

Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease

Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR–DMP323 complex, and distinctively for PRD25N–DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer–inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism.

A Transcriptionally Inactive ATF2 Variant Drives Melanomagenesis.

Melanoma is one of the most lethal cutaneous malignancies, characterized by chemoresistance and a striking propensity to metastasize. The transcription factor ATF2 elicits oncogenic activities in melanoma, and its inhibition attenuates melanoma development. Here, we show that expression of a transcriptionally inactive form of Atf2 (Atf2(Δ8,9)) promotes development of melanoma in mouse models. Atf2(Δ8,9)-driven tumors show enhanced pigmentation, immune infiltration, and metastatic propensity. Similar to mouse Atf2(Δ8,9), we have identified a transcriptionally inactive human ATF2 splice variant 5 (ATF2(SV5)) that enhances the growth and migration capacity of cultured melanoma cells and immortalized melanocytes. ATF2(SV5) expression is elevated in human melanoma specimens and is associated with poor prognosis. These findings point to an oncogenic function for ATF2 in melanoma development that appears to be independent of its transcriptional activity.

Andexanet: Effectively Reversing Anticoagulation

Despite direct oral anticoagulants becoming a mainstay of anticoagulant therapy, the effective, timely, and safe reversal of their anticoagulant effect remains challenging. Emerging evidence attests that andexanet, a recombinant and inactive variant of native factor X (FXa), competitively inhibits and counteracts the anticoagulant effect of many inhibitors of native activated FXa.

Mitochondrial gamma carbonic anhydrases are required for complex I assembly and plant reproductive development.

Complex I of the mitochondrial electron transport chain (mETC) in plants contains an extra domain that is made up from proteins homologous to prokaryotic gamma-carbonic anhydrases (γCA). This domain has been suggested to participate in complex I assembly or to support transport of mitochondrial CO2 to the chloroplast. Here, we generated mutants lacking CA1 and CA2 - two out of three CA proteins in Arabidopsis thaliana. Double mutants were characterized at the developmental and physiological levels. Furthermore, the composition and activity of the mETC were determined, and mutated CA versions were used for complementation assays. Embryo development of double mutants was strongly delayed and seed development stopped before maturation. Mutant plants could only be rescued on sucrose media, showed severe stress symptoms and never produced viable seeds. By contrast, callus cultures were only slightly affected in growth. Complex I was undetectable in the double mutants, but complex II and complex IV were upregulated concomitant with increased oxygen consumption in mitochondrial respiration. Ectopic expression of inactive CA variants was sufficient to complement the mutant phenotype. Data indicate that CA proteins are structurally required for complex I assembly and that reproductive development is dependent on the presence of complex I.

Paroxysmal nocturnal haemoglobinuria: a case presenting with isolated thrombocytopenia

ABSTRACTParoxysmal nocturnal haemoglobinuria (PNH) is a rare disease characterized by intravascular haemolysis, haemoglobinuria and thrombosis. The present study reported PNH patients presenting with isolated thrombocytopenia. A 31-year-old male patient was referred to haematology outpatient clinic because of thrombocytopenia detection. Initially, there was no anaemia, but apparent haemolytic anaemia occurred in the patient within a year. The PNH was diagnosed by using fluorescently labelled inactive variant of the protein aerolysin test. Anaemia, jaundice, thrombosis, fatigue, dyspnoea, dark urine, abdominal or gastric pain, dysphagia and erectile dysfunction are primary symptoms of PNH, leading to diagnosis. This is an atypical PNH case presenting with isolated thrombocytopenia, which led to difficulty and delay in diagnosis.

Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of HIV-1 Protease

The HIV-1 Protease (PR) mediates its own release and promotes the proteolysis of the Gag-Pol polyprotein into mature structural and functional forms indispensable for viral maturation. Due to its central role in the virus life cycle, the HIV-1 PR has been a prominent target for clinical protease inhibitors for almost 25 years. However the short replication cycle of the virus together with the error-prone reverse transcriptase lead to the rapidly evolving selection of inhibitor-resistant mutations in HIV-1 PR. We use here high-pressure NMR spectroscopy to characterize the cooperativity of unfolding, in the absence and in the presence of a symmetric inhibitor, of the active mature PR, the inactive variant PRD25N and of the clinical multi-drug resistant PR20. We found that binding of the inhibitor drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep free-energy minimum. We show that in order to avoid this conformational trap, PR20 has evolved to exhibit a nearly ideal two-state unfolding transition. Our study highlights the malleability of the HIV-1 PR folding pathways and illustrates how selection for drug resistant mutations can lead to a major remodeling of the free-energy landscape.

A mouse model for a partially inactive obesity-associated human MC3R variant

We previously reported children homozygous for two MC3R sequence variants (C17A+G241A) have greater fat mass than controls. Here we show, using homozygous knock-in mouse models in which we replace murine Mc3r with wild-type human (MC3RhWT/hWT) and double-mutant (C17A+G241A) human (MC3RhDM/hDM) MC3R, that MC3RhDM/hDM have greater weight and fat mass, increased energy intake and feeding efficiency, but reduced length and fat-free mass compared with MC3RhWT/hWT. MC3RhDM/hDM mice do not have increased adipose tissue inflammatory cell infiltration or greater expression of inflammatory markers despite their greater fat mass. Serum adiponectin levels are increased in MC3RhDM/hDM mice and MC3RhDM/hDM human subjects. MC3RhDM/hDM bone- and adipose tissue-derived mesenchymal stem cells (MSCs) differentiate into adipocytes that accumulate more triglyceride than MC3RhWT/hWT MSCs. MC3RhDM/hDM impacts nutrient partitioning to generate increased adipose tissue that appears metabolically healthy. These data confirm the importance of MC3R signalling in human metabolism and suggest a previously-unrecognized role for the MC3R in adipose tissue development.

SUMOylation represses SnRK1 signaling in Arabidopsis

The SnRK1 protein kinase balances cellular energy levels in accordance with extracellular conditions and is thereby key for plant stress tolerance. In addition, SnRK1 has been implicated in numerous growth and developmental processes from seed filling and maturation to flowering and senescence. Despite its importance, the mechanisms that regulate SnRK1 activity are poorly understood. Here, we demonstrate that the SnRK1 complex is SUMOylated on multiple subunits and identify SIZ1 as the E3 Small Ubiquitin-like Modifier (SUMO) ligase responsible for this modification. We further show that SnRK1 is ubiquitinated in a SIZ1-dependent manner, causing its degradation through the proteasome. In consequence, SnRK1 degradation is deficient in siz1-2 mutants, leading to its accumulation and hyperactivation of SnRK1 signaling. Finally, SnRK1 degradation is strictly dependent on its activity, as inactive SnRK1 variants are aberrantly stable but recover normal degradation when expressed as SUMO mimetics. Altogether, our data suggest that active SnRK1 triggers its own SUMOylation and degradation, establishing a negative feedback loop that attenuates SnRK1 signaling and prevents detrimental hyperactivation of stress responses.