The Nuclear SUMO-Targeted Ubiquitin Quality Control Network Regulates the Dynamics of Cytoplasmic Stress Granules.

Exposure of cells to heat or oxidative stress causes misfolding of proteins. To avoid toxic protein aggregation, cells have evolved nuclear and cytosolic protein quality control (PQC) systems. In response to proteotoxic stress, cells also limit protein synthesis by triggering transient storage of mRNAs and RNA-binding proteins (RBPs) in cytosolic stress granules (SGs). We demonstrate that the SUMO-targeted ubiquitin ligase (StUbL) pathway, which is part of the nuclear proteostasis network, regulates SG dynamics. We provide evidence that inactivation of SUMO deconjugases under proteotoxic stress initiates SUMO-primed, RNF4-dependent ubiquitylation of RBPs that typically condense into SGs. Impairment of SUMO-primed ubiquitylation drastically delays SG resolution upon stress release. Importantly, the StUbL system regulates compartmentalization of an amyotrophic lateral sclerosis (ALS)-associated FUS mutant in SGs. We propose that the StUbL system functions as surveillance pathway for aggregation-prone RBPs in the nucleus, thereby linking the nuclear and cytosolic axis of proteotoxic stress response.

SUMO-specific proteases and isopeptidases of the SENP family at a glance.

The ubiquitin-related SUMO system controls many cellular signaling networks. In mammalian cells, three SUMO forms (SUMO1, SUMO2 and SUMO3) act as covalent modifiers of up to thousands of cellular proteins. SUMO conjugation affects cell function mainly by regulating the plasticity of protein networks. Importantly, the modification is reversible and highly dynamic. Cysteine proteases of the sentrin-specific protease (SENP) family reverse SUMO conjugation in mammalian cells. In this Cell Science at a Glance article and the accompanying poster, we will summarize how the six members of the mammalian SENP family orchestrate multifaceted deconjugation events to coordinate cell processes, such as gene expression, the DNA damage response and inflammation.

Single Muscle Fiber Proteomics Reveals Distinct Protein Changes in Slow and Fast Fibers during Muscle Atrophy.

Skeletal muscles are composed of heterogeneous collections of fibers with different metabolic profiles. With varied neuronal innervation and fiber-type compositions, each muscle fulfils specific functions and responds differently to stimuli and perturbations. We assessed individual fibers by mass spectrometry to dissect protein changes after loss of neuronal innervation due to section of the sciatic nerve in mice. This proteomics approach enabled us to quantify ∼600 proteins per individual soleus and EDL (extensor digitorum longus) muscle fiber. Expression of myosin heavy chain (MyHC) in individual fibers enabled clustering of specific fiber types; comparison of fibers from control and denervated muscles with the same MyHC expression revealed restricted regulation of a total of 240 proteins in type-I, -IIa, or -IIb fibers 7 days after denervation. The levels of several mitochondrial and proteasomal proteins were significantly altered, indicating rapid adaption of metabolic processes after denervation. Furthermore, we observed fiber-type-specific regulation of proteins involved in calcium ion binding and transport, such as troponins, parvalbumin, and ATP2A2, indicating marked remodeling of muscle contractility after denervation. This study provides novel insight into how different muscle fiber types remodel their proteomes during muscular atrophy.

Dynamics of zebrafish fin regeneration using a pulsed SILAC approach.

The zebrafish owns remarkable regenerative capacities allowing regeneration of several tissues, including the heart, liver, and brain. To identify protein dynamics during fin regeneration we used a pulsed SILAC approach that enabled us to detect the incorporation of (13) C6 -lysine (Lys6) into newly synthesized proteins. Samples were taken at four different time points from noninjured and regrowing fins and incorporation rates were monitored using a combination of single-shot 4-h gradients and high-resolution tandem MS. We identified more than 5000 labeled proteins during the first 3 weeks of fin regeneration and were able to monitor proteins that are responsible for initializing and restoring the shape of these appendages. The comparison of Lys6 incorporation rates between noninjured and regrowing fins enabled us to identify proteins that are directly involved in regeneration. For example, we observed increased incorporation rates of two actinodin family members at the actinotrichia, which is a hairlike fiber structure at the tip of regrowing fins. Moreover, we used quantitative real-time RNA measurements of several candidate genes, including osteoglycin, si:ch211-288h17.3, and prostaglandin reductase 1 to correlate the mRNA expression to Lys6 incorporation data. This novel pulsed SILAC methodology in fish can be used as a versatile tool to monitor newly synthesized proteins and will help to characterize protein dynamics during regenerative processes in zebrafish beyond fin regeneration.

Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis.

Ribosome biogenesis is a multi-step process, in which a network of trans-acting factors ensures the coordinated assembly of pre-ribosomal particles in order to generate functional ribosomes. Ribosome biogenesis is tightly coordinated with cell proliferation and its perturbation activates a p53-dependent cell-cycle checkpoint. How p53-independent signalling networks connect impaired ribosome biogenesis to the cell-cycle machinery has remained largely enigmatic. We demonstrate that inactivation of the nucleolar SUMO isopeptidases SENP3 and SENP5 disturbs distinct steps of 40S and 60S ribosomal subunit assembly pathways, thereby triggering the canonical p53-dependent impaired ribosome biogenesis checkpoint. However, inactivation of SENP3 or SENP5 also induces a p53-independent checkpoint that converges on the specific downregulation of the key cell-cycle regulator CDK6. We further reveal that impaired ribosome biogenesis generally triggers the downregulation of CDK6, independent of the cellular p53 status. Altogether, these data define the role of SUMO signalling in ribosome biogenesis and unveil a p53-independent checkpoint of impaired ribosome biogenesis.

The SUMO Isopeptidase SENP6 Functions as a Rheostat of Chromatin Residency in Genome Maintenance and Chromosome Dynamics.

Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.

Skeletal Muscle-Specific Methyltransferase METTL21C Trimethylates p97 and Regulates Autophagy-Associated Protein Breakdown.

Protein aggregates and cytoplasmic vacuolization are major hallmarks of multisystem proteinopathies (MSPs) that lead to muscle weakness. Here, we identify METTL21C as a skeletal muscle-specific lysine methyltransferase. Insertion of a ß-galactosidase cassette into the Mettl21c mouse locus revealed that METTL21C is specifically expressed in MYH7-positive skeletal muscle fibers. Ablation of the Mettl21c gene reduced endurance capacity and led to age-dependent accumulation of autophagic vacuoles in skeletal muscle. Denervation-induced muscle atrophy highlighted further impairments of autophagy-related proteins, including LC3, p62, and cathepsins, in Mettl21c-/- muscles. In addition, we demonstrate that METTL21C interacts with the ATPase p97 (VCP), which is mutated in various human MSP conditions. We reveal that METTL21C trimethylates p97 on the Lys315 residue and found that loss of this modification reduced p97 hexamer formation and ATPase activity in vivo. We conclude that the methyltransferase METTL21C is an important modulator of protein degradation in skeletal muscle under both normal and enhanced protein breakdown conditions.