Photoacoustic Calorimetry Studies of O2-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain.

FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from Sinorhizobium meliloti and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for SmFixL*WT and the five variant proteins (SmFixL*R200H, SmFixL*R200Q, SmFixL*R200E, SmFixL*R200A, and SmFixL*I209M) with four intermediates from <20 ns to ∼1.5 µs associated with the photodissociation of CO from the heme. The altered thermodynamic profiles of the full-length SmFixL* variant proteins confirm that the conserved heme domain residues R200 and I209 are important for signal transduction. In contrast, the truncated heme domain, SmFixLH128-264, shows only a single, fast monophasic relaxation at <50 ns associated with the fast disruption of a salt bridge and release of CO to the solvent, suggesting that the full-length protein is necessary to observe the conformational changes that propagate the signal from the heme domain to the kinase domain.

Cell-Permeable Activity-Based Ubiquitin Probes Enable Intracellular Profiling of Human Deubiquitinases.

Advancement in our knowledge of deubiquitinases (DUBs) and their biological functions requires biochemical tools permitting interrogation of DUB activities under physiologically relevant conditions. Activity-based DUB probes (DUB ABPs) have been widely used in investigating the function and activity of DUBs. However, most ubiquitin (Ub)-based DUB ABPs are not cell-permeable, limiting their utility to purified proteins and cell lysates. Lysis of cells usually leads to dilution of the cytoplasm and disruption of the normal cellular organization, which may alter the activity of many DUBs and DUB complexes. Here, we report a new class of cell-permeable DUB ABPs that enable intracellular DUB profiling. We used a semisynthetic approach to generate modular ubiquitin-based DUB probes containing a reactive warhead for covalent trapping of DUBs with a catalytic cysteine. We employed cell-penetrating peptides (CPPs), particualrly cyclic polyarginine (cR10), to deliver the DUB ABPs into cells, as confirmed using live-cell fluorescence microscopy and DUB ABPs containing a fluorophore at the C-terminus of Ub. In comparison to TAT, enhanced intacellular delivery was observed through conjugation of a cyclic polyarginine (cR10) to the N-terminus of ubiquitin via a disulfide linkage. Using the new cell-permeable DUB ABPs, we carried out DUB profiling in intact HeLa cells, and identified active DUBs using immunocapture and label-free quantitative mass spectrometry. Additionally, we demonstrated that the cell-permeable DUB ABPs can be used in assessing the inhibition of DUBs by small-molecule inhibitors in intact cells. Our results indicate that cell-permeable DUB ABPs hold great promise in providing a better understanding of the cellular functions of DUBs and advancing drug discovery efforts targeting human DUBs.

Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors.

The deubiquitinases, or DUBs, are associated with various human diseases, including neurological disorders, cancer, and viral infection, making them excellent candidates for pharmacological intervention. Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are largely limited to recombinantly purified DUBs. Many DUBs are large multidomain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of recombinant protein generation and also recapitulates a physiologically relevant environment is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible, DUBs. Here, we report a cell lysate DUB assay based on AlphaLISA technology for high throughput screening. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. The assay was validated and adapted to a 1536-well format, which enabled a screening against UCHL1 as proof of principle using a library of 15 000 compounds. We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes.

Detailed analysis of the human mitochondrial contact site complex indicate a hierarchy of subunits.

Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components.

A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses.

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

C1orf163/RESA1 is a novel mitochondrial intermembrane space protein connected to respiratory chain assembly.

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60kDa and 150kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).

Requirements for the import of neisserial Omp85 into the outer membrane of human mitochondria.

ß-Barrel proteins are present only in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria. Fungal mitochondria were shown to readily import and assemble bacterial ß-barrel proteins, but human mitochondria exhibit certain selectivity. Whereas enterobacterial ß-barrel proteins are not imported, neisserial ones are. Of those, solely neisserial Omp85 is integrated into the outer membrane of mitochondria. In this study, we wanted to identify the signal that targets neisserial ß-barrel proteins to mitochondria. We exchanged parts of neisserial Omp85 and PorB with their Escherichia coli homologues BamA and OmpC. For PorB, we could show that its C-terminal quarter can direct OmpC to mitochondria. In the case of Omp85, we could identify several amino acids of the C-terminal ß-sorting signal as crucial for mitochondrial targeting. Additionally, we found that at least two POTRA (polypeptide-transport associated) domains and not only the ß-sorting signal of Omp85 are needed for its membrane integration and function in human mitochondria. We conclude that the signal that directs neisserial ß-barrel proteins to mitochondria is not conserved between these proteins. Furthermore, a linear mitochondrial targeting signal probably does not exist. It is possible that the secondary structure of ß-barrel proteins plays a role in directing these proteins to mitochondria.

Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes.

Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sorting and assembly machinery (SAM) necessary for the assembly of ß-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins mitofilin and CHCHD3, which we term the mitochondrial intermembrane space bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observed deficiency in the assembly of respiratory chain complexes. Long-term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondrially encoded subunits, pointing to a connection between the structural integrity of cristae, assembly of respiratory complexes, and/or the maintenance of mitochondrial DNA (mtDNA).

Neisserial Omp85 protein is selectively recognized and assembled into functional complexes in the outer membrane of human mitochondria.

As a consequence of their bacterial origin, mitochondria contain ß-barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the ß-barrel assembly machinery (BAM) required for biogenesis of ß-barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial ß-barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only ß-barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial ß-barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of ß-barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of ß-barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.