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The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing β-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including β2, β3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3-α3 and progress to β5 and β4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

BackgroundThe hydrophobic pocket (HP) of HIV-1 glycoprotein-41 ectodomain is defined by two chains of the N-heptad repeat trimer, within the protein-protein interface that mediates 6HB formation. It is a potential target for inhibitors of viral fusion, but its hydrophobic nature and proximity to membrane in situ has precluded ready analysis of inhibitor interactions.MethodsWe evaluated the sensitivity of 19F NMR and fluorescence for detecting peptide and small molecule binding to the HP and explored the effect of non-denaturing detergent or phospholipid as cosolvents and potential mimics of the membrane environment surrounding gp41.ResultsChemical shifts of aromatic fluorines were found to be sensitive to changes in the hydrogen bonding network that occurred when inhibitors transitioned from solvent into the HP or into ordered detergent micelles. Fluorescence intensities and emission maxima of autofluorescent compounds responded to changes in the local environment.ConclusionsGp41 - ligand binding occurred under all conditions, but was diminished in the presence of detergents. NMR and fluorescence studies revealed that dodecylphosphocholine (DPC) was a poor substitute for membrane in this system, while liposomes could mimic the membrane surroundings.General significanceOur findings suggest that development of high potency small molecule binders to the HP may be frustrated by competition between binding to the HP and binding to the bilayer membrane.

Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4E-BP1 is a key node in the regulation of protein synthesis, as activated 4E-BP1 represses global protein translation. Overexpression of 4E-BP1 mediates the benefits of dietary restriction and can counter metabolic stress, and 4E-BP1 disinhibition on mTORC1 repression may be neuroprotective; however, whether 4E-BP1 overexpression is neuroprotective in mammalian neurons is yet to be fully explored. To address this question, we generated 4E-BP1-overexpressing transgenic mice and confirmed marked reductions in protein translation in 4E-BP1-overexpressing primary neurons. After documenting that 4E-BP1-overexpressing neurons are resistant to proteotoxic stress elicited by brefeldin A treatment, we exposed primary neurons to three different Parkinson's disease (PD)-linked toxins (rotenone, maneb, or paraquat) and documented significant protection in neurons from newborn male and female 4E-BP1-OE transgenic mice. We observed 4E-BP1-dependent upregulation of genes encoding proteins that comprise the mitochondrial unfolded protein response, and noted 4E-BP1 overexpression required activation of the mitochondrial unfolded protein response for neuroprotection against rotenone toxicity. We also tested whether 4E-BP1 could prevent α-synuclein neurotoxicity by treating 4E-BP1-overexpressing primary neurons with α-synuclein preformed fibrils, and we observed marked reductions in α-synuclein aggregation and neurotoxicity, thus validating that 4E-BP1 is a powerful suppressor of PD-linked pathogenic insults. Our results indicate that increasing 4E-BP1 expression or enhancing 4E-BP1 activation can robustly induce the mitochondrial unfolded protein response and thus could be an appealing strategy for treating a variety of neurodegenerative diseases, including especially PD.SIGNIFICANCE STATEMENT In neurodegenerative disease, misfolded proteins accumulate and overwhelm normal systems of homeostasis and quality control. One mechanism for improving protein quality control is to reduce protein translation. Here we investigated whether neuronal overexpression of 4E-BP1, a key repressor of protein translation, can protect against misfolded protein stress and toxicities linked to Parkinson's disease, and found that 4E-BP1 overexpression prevented cell death in neurons treated with brefeldin A, rotenone, maneb, paraquat, or preformed fibrils of α-synuclein. When we sought the basis for 4E-BP1 neuroprotection, we discovered that 4E-BP1 activation promoted the mitochondrial unfolded protein response. Our findings highlight 4E-BP1 as a therapeutic target in neurodegenerative disease and underscore the importance of the mitochondrial unfolded protein response in neuroprotection against various insults.

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop-substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

Following assembly, the anthrax protective antigen (PA) forms an oligomeric translocon that unfolds and translocates either its lethal factor (LF) or edema factor (EF) into the host cell. Here, we report the cryo-EM structures of heptameric PA channels with partially unfolded LF and EF at 4.6 and 3.1-Å resolution, respectively. The first α helix and β strand of LF and EF unfold and dock into a deep amphipathic cleft, called the α clamp, which resides at the interface of two PA monomers. The α-clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins. EF undergoes a largescale conformational rearrangement when forming the complex with the channel. A critical loop in the PA binding interface is displaced for about 4 Å, leading to the weakening of the binding interface prior to translocation. These structures provide key insights into the molecular mechanisms of translocation-coupled protein unfolding and translocation.

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

Inorganic polyphosphate (polyP) constitutes one of the most conserved and ubiquitous molecules in biology. Recent work in bacteria demonstrated that polyP increases oxidative stress resistance by preventing stress-induced protein aggregation and promotes biofilm formation by stimulating functional amyloid formation. To gain insights into these two seemingly contradictory functions of polyP, we investigated the effects of polyP on the folding model lactate dehydrogenase. We discovered that the presence of polyP during the thermal unfolding process stabilizes folding intermediates of lactate dehydrogenase as soluble micro-β-aggregates with amyloid-like properties. Size and heterogeneity of the oligomers formed in this process were dependent on the polyP chain length, with longer chains forming smaller, more homogenous complexes. This ability of polyP to stabilize thermally unfolded proteins even upon exposure to extreme temperatures appears to contribute to the observed resistance of uropathogenic Escherichia coli toward severe heat shock treatment. These results suggest that the working mechanism of polyP is the same for both soluble and amyloidogenic proteins, with the ultimate outcome likely being determined by a combination of polyP chain length and the client protein itself. They help to explain how polyP can simultaneously function as general stress-protective chaperone and instigator of amyloidogenic processes in vivo.

In cyanobacteria, the KaiABC posttranslational oscillator drives circadian rhythms of gene expression and controls the timing of cell division. The Kai-based oscillator can be reconstituted in vitro, demonstrating that the clock can run without protein synthesis and degradation; however, protein degradation is known to be important for clock function in vivo. Here, we report that strains deficient in the ClpXP1P2 protease have, in addition to known long-period circadian rhythms, an exaggerated ability to synchronize with the external environment (reduced "jetlag") compared with WT strains. Deletion of the ClpX chaperone, but not the protease subunits ClpP1 or ClpP2, results in cell division defects in a manner that is dependent on the expression of a dusk-peaking factor. We propose that chaperone activities of ClpX are required to coordinate clock control of cell division whereas the protease activities of the ClpXP1P2 complex are required to maintain appropriate periodicity of the clock and its synchronization with the external environment.

Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10-15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and Tm ≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.

The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.

While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the Tm and Cm. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.

Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed "closure motifs". The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain-closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X-ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31comet We show that p31comet binding to the TRIP13 N-terminal domain positions the disordered MAD2 N-terminus for engagement by the TRIP13 "pore loops", which then unfold MAD2 in the presence of ATP N-terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13-mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain-closure motif complexes by TRIP13.

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function.

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.

The rational and predictable enhancement of protein stability is an important goal in protein design. Most efforts target the folded state, however stability is the free energy difference between the folded and unfolded states thus both are suitable targets. Strategies directed at the unfolded state usually seek to decrease chain entropy by introducing cross-links or by replacing glycines. Cross-linking has led to mixed results. Replacement of glycine with an l-amino acid, while reducing the entropy of the unfolded state, can introduce unfavorable steric interactions in the folded state, since glycine is often found in conformations that require a positive φ angle such as helical C-capping motifs or type I' and II″ β-turns. l-Amino acids are strongly disfavored in these conformations, but d-amino acids are not. However, there are few reported examples and conflicting results have been obtained when glycines are replaced with d-Ala. We critically examine the effect of Gly-to-d-Ala substitutions on protein stability using experimental approaches together with molecular dynamics simulations and free energy calculations. The data, together with a survey of high resolution structures, show that the vast majority of proteins can be stabilized by substitution of C-capping glycines with d-Ala. Sites suitable for substitutions can be identified via sequence alignment with a high degree of success. Steric clashes in the native state due to the new side chain are rarely observed, but are likely responsible for the destabilizing or null effect observed for the small subset of Gly-to-d-Ala substitutions which are not stabilizing. Changes in backbone solvation play less of a role. Favorable candidates for d-Ala substitution can be identified using a rapid algorithm based on molecular mechanics.

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.

ATP-dependent proteases of the AAA+ family, including Escherichia coli ClpXP and the eukaryotic proteasome, contribute to maintenance of cellular proteostasis. ClpXP unfolds and translocates substrates into an internal degradation chamber, using cycles of alternating dwell and burst phases. The ClpX motor performs chemical transformations during the dwell and translocates the substrate in increments of 1-4 nm during the burst, but the processes occurring during these phases remain unknown. Here we characterized the complete mechanochemical cycle of ClpXP, showing that ADP release and ATP binding occur nonsequentially during the dwell, whereas ATP hydrolysis and phosphate release occur during the burst. The highly conserved translocating loops within the ClpX pore are optimized to maximize motor power generation, the coupling between chemical and mechanical tasks, and the efficiency of protein processing. Conformational resetting of these loops between consecutive bursts appears to determine ADP release from individual ATPase subunits and the overall duration of the motor's cycle.

The extent of charging resulting from electrothermal supercharging for protein ions formed from various buffered aqueous solutions using nanoESI emitters with tip diameters between ∼1.5 μm and ∼310 nm is compared. Charging increases with decreasing tip size for proteins that are positively charged in solution but not for proteins that are negatively charged in solution. These results suggest that Coulombic attraction between positively charged protein molecules and the negatively charged glass surfaces in the tips of the emitters causes destabilization and even unfolding of proteins prior to nanoESI. Coulombic attraction to the negatively charged glass surfaces does not occur for negatively charged proteins and the extent of charging with electrothermal supercharging decreases with decreasing tip size. Smaller droplets are formed with smaller tips, and these droplets have shorter lifetimes for protein unfolding with electrothermal supercharging to occur prior to gaseous ion formation. Results from this study demonstrate simple principles to consider in order to optimize the extent of charging obtained with electrothermal supercharging, which should be useful for obtaining more structural information in tandem mass spectrometry.

Platelet activation in blood flow under high, overcritical shear rates is initiated by Von Willebrand factor. Despite the large amount of experimental data that have been obtained, the value of the critical shear rate, above which von Willebrand factor starts to activate platelets, is still controversial. Here, we recommend a theoretical approach to elucidate how the critical blood shear rate is dependent on von Willebrand factor size. We derived a diagram of platelet activation according to the shear rate and von Willebrand factor multimer size. We succeeded in deriving an explicit formula for the dependence of the critical shear rate on von Willebrand factor molecule size. The platelet activation risk index was introduced. This index is dependent on the flow conditions, number of monomers in von Willebrand factor, and platelet sensitivity. Probable medical applications of the platelet activation risk index as a universal prognostic index are discussed.

The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.

In vitro, replacing KCl with potassium glutamate (KGlu), the Escherichia coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu- in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to the effects of other stabilizers (KCl, KF, and K2SO4) and destabilizers (GuHCl and GuHSCN). GuHSCN titrations at 20 ° C, performed as a function of the concentration of KGlu or another salt and monitored by NTL9 fluorescence, are analyzed to obtain R-values quantifying the Hofmeister salt concentration (m3) dependence of the unfolding equilibrium constant K(obs) [r-value = −d ln K(obs)/dm3 = (1/RT) dΔG(obs) ° /dm3 = m-value/RT]. r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 ° C, we determine a KGlu r-value of 3.22 m(−1) and K2SO4, KF, KCl, GuHCl, and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38, and −3.00 m(−1), respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 ° C.

Unusually large von Willebrand factor (VWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease, two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding affinity is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to His(1322) across the β2-β3 hairpin in the GPIbα binding interface, which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

Unusually large von Willebrand factor (VWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease, two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding affinity is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to His(1322) across the β2-β3 hairpin in the GPIbα binding interface, which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

The tumor suppressor p53 plays a unique role as a central hub of numerous cell proliferation and apoptotic pathways, and its malfunction due to mutations is a major cause of various malignancies. Therefore, it serves as an attractive target for developing novel anticancer therapeutics. Because of its intrinsically unstable DNA binding domain, p53 unfolds rapidly at physiological temperature. Certain mutants shift the equilibrium toward the unfolded state and yield high-molecular weight, nonfunctional, and cytotoxic β-sheet-rich aggregates that share tinctorial and conformational similarities with amyloid deposits found in various protein misfolding diseases. Here, we examined the effect of a novel protein assembly modulator, the lysine (Lys)-specific molecular tweezer, CLR01, on different aggregation stages of misfolded mutant p53 in vitro and on the cytotoxicity of the resulting p53 aggregates in cell culture. We found that CLR01 induced rapid formation of β-sheet-rich, intermediate-size p53 aggregates yet inhibited further p53 aggregation and reduced the cytotoxicity of the resulting aggregates. Our data suggest that aggregation modulators, such as CLR01, could prevent the formation of toxic p53 aggregates.

The tumor suppressor p53 plays a unique role as a central hub of numerous cell proliferation and apoptotic pathways, and its malfunction due to mutations is a major cause of various malignancies. Therefore, it serves as an attractive target for developing novel anticancer therapeutics. Because of its intrinsically unstable DNA binding domain, p53 unfolds rapidly at physiological temperature. Certain mutants shift the equilibrium toward the unfolded state and yield high-molecular weight, nonfunctional, and cytotoxic β-sheet-rich aggregates that share tinctorial and conformational similarities with amyloid deposits found in various protein misfolding diseases. Here, we examined the effect of a novel protein assembly modulator, the lysine (Lys)-specific molecular tweezer, CLR01, on different aggregation stages of misfolded mutant p53 in vitro and on the cytotoxicity of the resulting p53 aggregates in cell culture. We found that CLR01 induced rapid formation of β-sheet-rich, intermediate-size p53 aggregates yet inhibited further p53 aggregation and reduced the cytotoxicity of the resulting aggregates. Our data suggest that aggregation modulators, such as CLR01, could prevent the formation of toxic p53 aggregates.

Long-term dietary supplementation with resveratrol protects against cardiovascular disease, osteoporesis, and metabolic decline. This study determined how long-term dietary resveratrol treatment protects against retinal ganglion cell (RGC) dendrite loss after optic nerve injury and alters the resolution of the unfolded protein response. Associated changes in markers of endoplasmic reticulum stress in RGCs also were investigated. Young-adult Thy1-yellow fluorescent protein (YFP) and C57BL/6 mice received either control diet or diet containing resveratrol for approximately 1 year. Both groups then received optic nerve crush (ONC). Fluorescent RGC dendrites in the Thy1-YFP mice were imaged weekly for 4 weeks after ONC. There was progressive loss of dendrite length in all RGC types within the mice that received control diet. Resveratrol delayed loss of dendrite complexity and complete dendrite loss for most RGC types. However, there were variations in the rate of retraction among different RGC types. Three weeks after ONC, cytoplasmic binding immunoglobulin protein (BiP) suppression observed in control diet ganglion cell layer neurons was reversed in mice that received resveratrol, nuclear C/EBP homologous protein (CHOP) was near baseline in control diet eyes but was moderately increased by resveratrol; and increased nuclear X-box-binding protein-1 (XBP-1) observed in control diet eyes was reduced in eyes that received resveratrol to the same level as in control diet uncrushed eyes. These results indicate that protection of dendrites by resveratrol after ONC differs among RGC types and suggest that alterations in long-term expression of binding immunoglobulin protein, CHOP, and XBP-1 may contribute to the resveratrol-mediated protection of RGC dendrites after ONC.

Long-term dietary supplementation with resveratrol protects against cardiovascular disease, osteoporesis, and metabolic decline. This study determined how long-term dietary resveratrol treatment protects against retinal ganglion cell (RGC) dendrite loss after optic nerve injury and alters the resolution of the unfolded protein response. Associated changes in markers of endoplasmic reticulum stress in RGCs also were investigated. Young-adult Thy1-yellow fluorescent protein (YFP) and C57BL/6 mice received either control diet or diet containing resveratrol for approximately 1 year. Both groups then received optic nerve crush (ONC). Fluorescent RGC dendrites in the Thy1-YFP mice were imaged weekly for 4 weeks after ONC. There was progressive loss of dendrite length in all RGC types within the mice that received control diet. Resveratrol delayed loss of dendrite complexity and complete dendrite loss for most RGC types. However, there were variations in the rate of retraction among different RGC types. Three weeks after ONC, cytoplasmic binding immunoglobulin protein (BiP) suppression observed in control diet ganglion cell layer neurons was reversed in mice that received resveratrol, nuclear C/EBP homologous protein (CHOP) was near baseline in control diet eyes but was moderately increased by resveratrol; and increased nuclear X-box-binding protein-1 (XBP-1) observed in control diet eyes was reduced in eyes that received resveratrol to the same level as in control diet uncrushed eyes. These results indicate that protection of dendrites by resveratrol after ONC differs among RGC types and suggest that alterations in long-term expression of binding immunoglobulin protein, CHOP, and XBP-1 may contribute to the resveratrol-mediated protection of RGC dendrites after ONC.

We apply novel atomistic simulations based on potential energy surface exploration to investigate the constant force-induced unfolding of ubiquitin. At the experimentally-studied force clamping level of 100 pN, we find a new unfolding mechanism starting with the detachment between β5 and β3 involving the binding site of ubiquitin, the Ile44 residue. This new unfolding pathway leads to the discovery of new intermediate configurations, which correspond to the end-to-end extensions previously seen experimentally. More importantly, it demonstrates the novel finding that the binding site of ubiquitin can be responsible not only for its biological functions, but also its unfolding dynamics. We also report in contrast to previous single molecule constant force experiments that when the clamping force becomes smaller than about 300 pN, the number of intermediate configurations increases dramatically, where almost all unfolding events at 100 pN involve an intermediate configuration. By directly calculating the life times of the intermediate configurations from the height of the barriers that were crossed on the potential energy surface, we demonstrate that these intermediate states were likely not observed experimentally due to their lifetimes typically being about two orders of magnitude smaller than the experimental temporal resolution.

We apply novel atomistic simulations based on potential energy surface exploration to investigate the constant force-induced unfolding of ubiquitin. At the experimentally-studied force clamping level of 100 pN, we find a new unfolding mechanism starting with the detachment between β5 and β3 involving the binding site of ubiquitin, the Ile44 residue. This new unfolding pathway leads to the discovery of new intermediate configurations, which correspond to the end-to-end extensions previously seen experimentally. More importantly, it demonstrates the novel finding that the binding site of ubiquitin can be responsible not only for its biological functions, but also its unfolding dynamics. We also report in contrast to previous single molecule constant force experiments that when the clamping force becomes smaller than about 300 pN, the number of intermediate configurations increases dramatically, where almost all unfolding events at 100 pN involve an intermediate configuration. By directly calculating the life times of the intermediate configurations from the height of the barriers that were crossed on the potential energy surface, we demonstrate that these intermediate states were likely not observed experimentally due to their lifetimes typically being about two orders of magnitude smaller than the experimental temporal resolution.

Exposure of cells to reactive oxygen species (ROS) causes a rapid and significant drop in intracellular ATP levels. This energy depletion negatively affects ATP-dependent chaperone systems, making ROS-mediated protein unfolding and aggregation a potentially very challenging problem. Here we show that Get3, a protein involved in ATP-dependent targeting of tail-anchored (TA) proteins under nonstress conditions, turns into an effective ATP-independent chaperone when oxidized. Activation of Get3's chaperone function, which is a fully reversible process, involves disulfide bond formation, metal release, and its conversion into distinct, higher oligomeric structures. Mutational studies demonstrate that the chaperone activity of Get3 is functionally distinct from and likely mutually exclusive with its targeting function, and responsible for the oxidative stress-sensitive phenotype that has long been noted for yeast cells lacking functional Get3. These results provide convincing evidence that Get3 functions as a redox-regulated chaperone, effectively protecting eukaryotic cells against oxidative protein damage.

Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pKa measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.

Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasomes capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.

Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.

Approximately 10% of water-soluble proteins are considered kinetically stable with unfolding half-lives in the range of weeks to millenia. These proteins only rarely sample the unfolded state and may never unfold on their respective biological time scales. It is still not known whether membrane proteins can be kinetically stable, however. Here we examine the subunit dissociation rate of the trimeric membrane enzyme, diacylglycerol kinase, from Escherichia coli as a proxy for complete unfolding. We find that dissociation occurs with a half-life of at least several weeks, demonstrating that membrane proteins can remain locked in a folded state for long periods of time. These results reveal that evolution can use kinetic stability to regulate the biological function of membrane proteins, as it can for soluble proteins. Moreover, it appears that the generation of kinetic stability could be a viable target for membrane protein engineering efforts.

BACKGROUND: ATP-dependent proteases translocate and unfold their substrates. RESULTS: A human virus sequence with only Gly and Ala residues causes similar dysfunctions of eukaryotic and prokaryotic protease motors: unfolding failure. CONCLUSION: Sequences with amino acids of simple shape and small size impair unfolding of contiguous stable domains. SIGNIFICANCE: Compartmented ATP-dependent proteases of diverse origin share conserved principles of interaction between translocase/effector and substrate/recipient. ATP-dependent proteases engage, translocate, and unfold substrate proteins. A sequence with only Gly and Ala residues (glycine-alanine repeat; GAr) encoded by the Epstein-Barr virus of humans inhibits eukaryotic proteasome activity. It causes the ATPase translocase to slip on its protein track, stalling unfolding and interrupting degradation. The bacterial protease ClpXP is structurally simpler than the proteasome but has related elements: a regulatory ATPase complex (ClpX) and associated proteolytic chamber (ClpP). In this study, GAr sequences were found to impair ClpXP function much as in proteasomes. Stalling depended on interaction between a GAr and a suitably spaced and positioned folded domain resistant to mechanical unfolding. Persistent unfolding failure results in the interruption of degradation and the production of partial degradation products that include the resistant domain. The capacity of various sequences to cause unfolding failure was investigated. Among those tested, a GAr was most effective, implying that viral selection had optimized processivity failure. More generally, amino acids of simple shape and small size promoted unfolding failure. The ClpX ATPase is a homohexamer. Partial degradation products could exit the complex through transient gaps between the ClpX monomers or, alternatively, by backing out. Production of intermediates by diverse topological forms of the hexamer was shown to be similar, excluding lateral escape. In principle, a GAr could interrupt degradation because 1) the translocase thrusts forward less effectively or because 2) the translocase retains substrate less well when resetting between forward strokes. Kinetic analysis showed that the predominant effect was through the second of these mechanisms.

BackgroundATP-dependent proteases translocate and unfold their substrates.ResultsA human virus sequence with only Gly and Ala residues causes similar dysfunctions of eukaryotic and prokaryotic protease motors: unfolding failure.ConclusionSequences with amino acids of simple shape and small size impair unfolding of contiguous stable domains.SignificanceCompartmented ATP-dependent proteases of diverse origin share conserved principles of interaction between translocase/effector and substrate/recipient. ATP-dependent proteases engage, translocate, and unfold substrate proteins. A sequence with only Gly and Ala residues (glycine-alanine repeat; GAr) encoded by the Epstein-Barr virus of humans inhibits eukaryotic proteasome activity. It causes the ATPase translocase to slip on its protein track, stalling unfolding and interrupting degradation. The bacterial protease ClpXP is structurally simpler than the proteasome but has related elements: a regulatory ATPase complex (ClpX) and associated proteolytic chamber (ClpP). In this study, GAr sequences were found to impair ClpXP function much as in proteasomes. Stalling depended on interaction between a GAr and a suitably spaced and positioned folded domain resistant to mechanical unfolding. Persistent unfolding failure results in the interruption of degradation and the production of partial degradation products that include the resistant domain. The capacity of various sequences to cause unfolding failure was investigated. Among those tested, a GAr was most effective, implying that viral selection had optimized processivity failure. More generally, amino acids of simple shape and small size promoted unfolding failure. The ClpX ATPase is a homohexamer. Partial degradation products could exit the complex through transient gaps between the ClpX monomers or, alternatively, by backing out. Production of intermediates by diverse topological forms of the hexamer was shown to be similar, excluding lateral escape. In principle, a GAr could interrupt degradation because 1) the translocase thrusts forward less effectively or because 2) the translocase retains substrate less well when resetting between forward strokes. Kinetic analysis showed that the predominant effect was through the second of these mechanisms.

BackgroundATP-dependent proteases translocate and unfold their substrates.ResultsA human virus sequence with only Gly and Ala residues causes similar dysfunctions of eukaryotic and prokaryotic protease motors: unfolding failure.ConclusionSequences with amino acids of simple shape and small size impair unfolding of contiguous stable domains.SignificanceCompartmented ATP-dependent proteases of diverse origin share conserved principles of interaction between translocase/effector and substrate/recipient. ATP-dependent proteases engage, translocate, and unfold substrate proteins. A sequence with only Gly and Ala residues (glycine-alanine repeat; GAr) encoded by the Epstein-Barr virus of humans inhibits eukaryotic proteasome activity. It causes the ATPase translocase to slip on its protein track, stalling unfolding and interrupting degradation. The bacterial protease ClpXP is structurally simpler than the proteasome but has related elements: a regulatory ATPase complex (ClpX) and associated proteolytic chamber (ClpP). In this study, GAr sequences were found to impair ClpXP function much as in proteasomes. Stalling depended on interaction between a GAr and a suitably spaced and positioned folded domain resistant to mechanical unfolding. Persistent unfolding failure results in the interruption of degradation and the production of partial degradation products that include the resistant domain. The capacity of various sequences to cause unfolding failure was investigated. Among those tested, a GAr was most effective, implying that viral selection had optimized processivity failure. More generally, amino acids of simple shape and small size promoted unfolding failure. The ClpX ATPase is a homohexamer. Partial degradation products could exit the complex through transient gaps between the ClpX monomers or, alternatively, by backing out. Production of intermediates by diverse topological forms of the hexamer was shown to be similar, excluding lateral escape. In principle, a GAr could interrupt degradation because 1) the translocase thrusts forward less effectively or because 2) the translocase retains substrate less well when resetting between forward strokes. Kinetic analysis showed that the predominant effect was through the second of these mechanisms.