Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation.

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

Ribosomal protein RPL26 is the principal target of UFMylation.

Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER.

Characterization of protein complexes of the endoplasmic reticulum-associated degradation E3 ubiquitin ligase Hrd1.

Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

A PDI family network acts distinctly and coordinately with ERp29 to facilitate polyomavirus infection.

Endoplasmic reticulum (ER)-to-cytosol membrane transport is a decisive infection step for the murine polyomavirus (Py). We previously determined that ERp29, a protein disulfide isomerase (PDI) member, extrudes the Py VP1 C-terminal arm to initiate ER membrane penetration. This reaction requires disruption of Py's disulfide bonds. Here, we found that the PDI family members ERp57, PDI, and ERp72 facilitate virus infection. However, while all three proteins disrupt Py's disulfide bonds in vitro, only ERp57 and PDI operate in concert with ERp29 to unfold the VP1 C-terminal arm. An alkylated Py cannot stimulate infection, implying a pivotal role of viral free cysteines during infection. Consistent with this, we found that although PDI and ERp72 reduce Py, ERp57 principally isomerizes the virus in vitro, a reaction that requires viral free cysteines. Our mutagenesis study subsequently identified VP1 C11 and C15 as important for infection, suggesting a role for these residues during isomerization. C11 and C15 also act together to stabilize interpentamer interactions for a subset of the virus pentamers, likely because some of these residues form interpentamer disulfide bonds. This study reveals how a PDI family functions coordinately and distinctly to promote Py infection and pinpoints a role of viral cysteines in this process.

4,6-Dinitro-N,N'-di-n-octylbenzene-1,3-diamine, 4,6-dinitro-N,N'-di-n-undecylbenzene-1,3-diamine and N,N'-bis(2,4-dinitrophenyl)octane-1,8-diamine.

4,6-Dinitro-N,N'-di-n-octylbenzene-1,3-diamine, C(22)H(38)N(4)O(4), (I), 4,6-dinitro-N,N'-di-n-undecylbenzene-1,3-diamine, C(28)H(50)N(4)O(4), (II), and N,N'-bis(2,4-dinitrophenyl)octane-1,8-diamine, C(20)H(24)N(6)O(8), (III), are the first synthetic meta-dinitroarenes functionalized with long-chain aliphatic amine groups to be structurally characterized. The intra- and intermolecular interactions in these model compounds provide information that can be used to help understand the physical properties of corresponding polymers with similar functionalities. Compounds (I) and (II) possess near-mirror symmetry, with the octyl and undecyl chains adopting fully extended anti conformations in the same direction with respect to the ring. Compound (III) rests on a center of inversion that occupies the mid-point of the central C-C bond of the octyl chain. The middle six C atoms of the chain form an anti arrangement, while the remaining two C atoms take hard turns almost perpendicular to the rest of the chain. All three molecules display intramolecular N-H...O hydrogen bonds between the amine and nitro groups, with the same NH group forming a bifurcated intermolecular hydrogen bond to the nitro O atom of an adjacent molecule. In each case, these interactions link the molecules into one-dimensional molecular chains. In (I) and (II), these chains pack so that the pendant alkyl groups are interleaved parallel to one another, maximizing nonbonded C-H contacts. In (III), the alkyl groups are more isolated within the molecular chains and the primary nonbonded contacts between the chains appear to involve the nitro groups not involved in the hydrogen bonding.

1,3-Bis(ethylamino)-2-nitrobenzene, 1,3-bis(n-octylamino)-2-nitrobenzene and 4-ethylamino-2-methyl-1H-benzimidazole.

1,3-Bis(ethylamino)-2-nitrobenzene, C(10)H(15)N(3)O(2), (I), and 1,3-bis(n-octylamino)-2-nitrobenzene, C(22)H(39)N(3)O(2), (II), are the first structurally characterized 1,3-bis(n-alkylamino)-2-nitrobenzenes. Both molecules are bisected though the nitro N atom and the 2-C and 5-C atoms of the ring by twofold rotation axes. Both display intramolecular N-H...O hydrogen bonds between the amine and nitro groups, but no intermolecular hydrogen bonding. The nearly planar molecules pack into flat layers ca 3.4 A apart that interact by hydrophobic interactions involving the n-alkyl groups rather than by pi-pi interactions between the rings. The intra- and intermolecular interactions in these molecules are of interest in understanding the physical properties of polymers made from them. Upon heating in the presence of anhydrous potassium carbonate in dimethylacetamide, (I) and (II) cyclize with formal loss of hydrogen peroxide to form substituted benzimidazoles. Thus, 4-ethylamino-2-methyl-1H-benzimidazole, C(10)H(13)N(3), (III), was obtained from (I) under these reaction conditions. Compound (III) contains two independent molecules with no imposed internal symmetry. The molecules are linked into chains via N-H...N hydrogen bonds involving the imidazole rings, while the ethylamino groups do not participate in any hydrogen bonding. This is the first reported structure of a benzimidazole derivative with 4-amino and 2-alkyl substituents.

N-(n-Dec-yl)-4-nitro-aniline.

N-(n-Dec-yl)-4-nitro-aniline, C(16)H(26)N(2)O(2), crystallizes with two essentially planar mol-ecules in the asymmetric unit. The decyl chains are fully extended in an anti conformation. The mol-ecules pack in planar layers, within which mol-ecules are linked into chains by two approximately linear N-H⋯O hydrogen bonds between the amine N atom and one O atom of the nitro group of an adjacent mol-ecule. These mol-ecular chains propagate via inter-leaving of the decyl chains to form the two dimensional sheets. The sheets are associated exclusively via non-bonded contacts. The structure has features in common with those of other N-alkyl-4-nitro-anilines, but also subtle differences in packing.

Endoplasmic reticulum-dependent redox reactions control endoplasmic reticulum-associated degradation and pathogen entry.

SIGNIFICANCE: Protein misfolding within the endoplasmic reticulum (ER) is managed by an ER quality control system that retro-translocates aberrant proteins into the cytosol for proteasomal destruction. This process, known as ER-associated degradation, utilizes the action of ER redox enzymes to accommodate the disulfide-bonded nature of misfolded proteins. Strikingly, various pathogenic viruses and toxins co-opt these redox components to reach the cytosol during entry. These redox factors thus regulate critical cellular homeostasis and host-pathogen interactions. RECENT ADVANCES: Recent studies identify specific members of the protein disulfide isomerase (PDI) family, which use their chaperone and catalytic activities, in engaging both misfolded ER proteins and pathogens. CRITICAL ISSUES: The precise molecular mechanism by which a dedicated PDI family member disrupts the disulfide bonds in the misfolded ER proteins and pathogens, as well as how they act to unfold these substrates to promote their ER-to-cytosol membrane transport, remain poorly characterized. FUTURE DIRECTIONS: How PDI family members distinguish folded versus misfolded ER substrates remains enigmatic. What physical characteristics surrounding a substrate's disulfide bond instruct PDI that it is mispaired or native? For the pathogens, as their disulfide bonds normally serve a critical role in providing physical support, what conformational changes experienced in the host enable their disulfide bonds to be disrupted? A combination of more rigorous biochemical and high-resolution structural studies should begin to address these questions.

High-throughput screen for Escherichia coli heat shock protein 70 (Hsp70/DnaK): ATPase assay in low volume by exploiting energy transfer.

Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate detection reagents, such as quinaldine red (QR). Although such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70's weak enzymatic activity have combined to create significant challenges in high-throughput screening. To overcome these difficulties, the authors have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal Biochem 2005;342:254-259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, the authors tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z' ~0.6, coefficient of variation ~8%), and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70 family members.