Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC.

Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.

Dual Role of Ribosome-Binding Domain of NAC as a Potent Suppressor of Protein Aggregation and Aging-Related Proteinopathies.

The nascent polypeptide-associated complex (NAC) is a conserved ribosome-associated protein biogenesis factor. Whether NAC exerts chaperone activity and whether this function is restricted to de novo protein synthesis is unknown. Here, we demonstrate that NAC directly exerts chaperone activity toward structurally diverse model substrates including polyglutamine (PolyQ) proteins, firefly luciferase, and Aß40. Strikingly, we identified the positively charged ribosome-binding domain in the N terminus of the ßNAC subunit (N-ßNAC) as a major chaperone entity of NAC. N-ßNAC by itself suppressed aggregation of PolyQ-expanded proteins in vitro, and the positive charge of this domain was critical for this activity. Moreover, we found that NAC also exerts a ribosome-independent chaperone function in vivo. Consistently, we found that a substantial fraction of NAC is non-ribosomal bound in higher eukaryotes. In sum, NAC is a potent suppressor of aggregation and proteotoxicity of mutant PolyQ-expanded proteins associated with human diseases like Huntington's disease and spinocerebellar ataxias.

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins.

Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3'-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.

Near-UV circular dichroism reveals structural transitions of vimentin subunits during intermediate filament assembly.

In vitro assembly of vimentin intermediate filaments (IFs) proceeds from soluble, reconstituted tetrameric complexes to mature filaments in three distinct stages: (1) within the first seconds after initiation of assembly, tetramers laterally associate into unit-length filaments (ULFs), on average 17 nm wide; (2) for the next few minutes, ULFs grow by longitudinal annealing into short, immature filaments; (3) almost concomitant with elongation, these immature filaments begin to radially compact, yielding approximately 11-nm-wide IFs at around 15 min. The near-UV CD signal of soluble tetramers exhibits two main peaks at 285 and 278 nm, which do not change during ULF formation. In contrast, the CD signal of mature IFs exhibits two major changes: (1) the 278-nm band, denoting the transition of the tyrosines from the ground state to the first vibrational mode of the excited state, is lost; (2) a red-shifted band appears at 291 nm, indicating the emergence of a new electronic species. These changes take place independently and at different time scales. The 278-nm signal disappears within the first minute of assembly, compatible with increased rigidity of the tyrosines during elongation of the ULFs. The rise of the 291-nm band has a lifetime of approximately 13 min and denotes the generation of phenolates by deprotonation of the tyrosines' hydroxyl group after they relocalize into a negatively charged environment. The appearance of such tyrosine-binding "pockets" in the assembling filaments highlights an essential part of the molecular rearrangements characterizing the later stages of the assembly process, including the radial compaction.

A pilot randomized controlled trial of renal protection with pioglitazone in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy progresses relentlessly to end-stage renal disease (ESRD). Animal experiments have found that peroxisome proliferator activated receptor-gamma (PPAR-gamma)-based therapy can have a glucose independent effect on renal protection. We hypothesized that PPAR-gamma-based antidiabetic therapy would result in greater reduction in proteinuria compared to sulfonylurea-based therapy. METHODS: In 44 patients with overt diabetic nephropathy, an open-label, blinded end point trial was conducted in which subjects were randomized to either pioglitazone or glipizide to achieve similar glucose control. Proteinuria was assessed by two collections of 24-hour urine samples each month for 4 months. RESULTS: The glipizide group had an adjusted mean increase in proteinuria of 6.1% (95% CI -11.7%, 23.8%), whereas the pioglitazone group had a reduction of 7.2% (95% CI -24.9%, 10.6%). The adjusted reduction with pioglitazone of 13.2% (95% CI -38.4%, 11.9%) was not statistically significant (P= 0.294). Baseline proteinuria, diastolic ambulatory blood pressure, and serum albumin concentration were independent predictors of reduction in proteinuria. The frequency and patterns of adverse events were similar in the two groups. CONCLUSION: In patients with advanced diabetic nephropathy, we found no reduction in proteinuria over 4 months. These data are useful to design larger studies with longer duration of follow-up to demonstrate renal protection of PPAR-gamma agonists.

Oxidative stress and renal injury with intravenous iron in patients with chronic kidney disease.

BACKGROUND: Intravenous iron is widely prescribed in patients with chronic kidney disease (CKD) and can cause oxidative stress. The relationship of oxidative stress and renal injury in patients with CKD is unknown. Whether renal injury can occur at a time point when transferrin is incompletely saturated is also unclear. METHODS: We conducted a randomized, open-label, parallel group trial to compare the oxidative stress induced by intravenous administration of 100 mg iron sucrose over 5 minutes and its protection with N-acetylcysteine (NAC) in 20 subjects with stage 3 or 4 CKD. Transferrin saturation was measured with urea polyacrylamide gel electrophoresis, oxidative stress by malondialdehyde (MDA) measurement by high-performance liquid chromatography, and renal injury by enzymuria and proteinuria. Reduced and oxidized glutathione and free radical scavengers as well as urinary monocyte chemoattractant protein-1 were also measured. RESULTS: Parenteral iron increased plasma concentration and urinary excretion rate of MDA, a biomarker of lipid peroxidation, within 15 to 30 minutes of iron sucrose administration. This was accompanied by enzymuria and increase in proteinuria. In contrast, saturation of transferrin was not maximally seen until 3 hours after the end of infusion. Oxidative stress, enzymuria and proteinuria were transient and were completely resolved in 24 hours. NAC reduced acute generation of systemic oxidative stress but failed to abrogate proteinuria or enzymuria. CONCLUSION: Intravenous iron produces oxidative stress that is associated with transient proteinuria and tubular damage. The rapid production of oxidative stress even when transferrin is not completely saturation suggests free iron independent mechanism(s) to be operative in producing oxidative stress and transient renal injury. Long-term implications of these findings need further study.