Summary It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate non-co-operatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits co-operatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable ‘cross-β’ cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and non-amyloid clients, which impose distinct mechanical demands.

SUMMARY The structural basis by which Hsp104 dissolves disordered aggregates and prions is unknown. A single subunit within the Hsp104 hexamer can solubilize disordered aggregates, whereas prion dissolution requires collaboration by multiple Hsp104 subunits. Here, we establish that the poorly understood Hsp104 N-terminal domain (NTD) enables this operational plasticity. Hsp104 lacking the NTD (Hsp104ΔN) dissolves disordered aggregates but cannot dissolve prions or be potentiated by activating mutations. We define how Hsp104ΔN invariably stimulates Sup35 prionogenesis by fragmenting prions without solubilizing Sup35, whereas Hsp104 couples Sup35 prion fragmentation and dissolution. Volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (hydrolysis transition state mimic), and ADP via small-angle X-ray scattering revealed a peristaltic pumping motion upon ATP hydrolysis, which drives directional substrate translocation through the central Hsp104 channel and is profoundly altered in Hsp104ΔN. We establish that the Hsp104 NTD enables cooperative substrate translocation, which is critical for prion dissolution and potentiated disaggregase activity.

The lactose (lac) repressor is an allosteric protein that can respond to environmental changes. Mutations introduced into the DNA binding domain and the effector binding pocket affect the repressor’s ability to respond to its environment. We have demonstrated how the observed phenotype is a consequence of altering the thermodynamic equilibrium constants. We discuss mutant repressors, which (1) show tighter repression; (2) induce with a previously noninducing species, orthonitrophenyl-β-d-galactoside; and (3) transform an inducible switch to one that is corepressed. The ability of point mutations to change multiple thermodynamic constants, and hence drastically alter the repressor’s phenotype, shows how allosteric proteins can perform a wide array of similar yet distinct functions such as that exhibited in the Lac/Gal family of bacterial repressors.

Alzheimer's disease (AD) is the most common form of dementia in the elderly. Classic symptoms of the disease include memory loss and confusion associated with the hallmark neuro-pathologic lesions of neurofibrillary tangles (NFT) and senile plaques (SP) and their sequelae, gray matter atrophy. Volumetric assessment methods measure tissue atrophy, which typically follows early biochemical changes. An alternate MRI contrast mechanism to visualize the early pathological changes is T 1ρ (or "T-1-rho"), the spin lattice relaxation time constant in the rotating frame, which determines the decay of the transverse magnetization in the presence of a "spin-lock" radio-frequency field. Macromolecular changes (in plaques and tangles) that accompany early AD are expected to alter bulk water T 1ρ relaxation times. In this work, we measure T 1ρ MRI on patients with clinically diagnosed AD, MCI and in age-matched ognitively normal control subjects in order to compare T 1ρ values with changes in brain volume in the same regions of the brain and demonstrate that T 1ρ can potentially constitute an important biomarker of AD.

Purpose: To develop a T1-prepared, balanced gradient echo (b-GRE) pulse sequence for rapid three-dimensional (3D) T1 relaxation mapping within the time constraints of a clinical exam (Ͻ10 minutes), examine the effect of acquisition on the measured T1 relaxation time and optimize 3D T1 pulse sequences for the knee joint and spine. Materials and Methods:A pulse sequence consisting of inversion recovery-prepared, fat saturation, T1-preparation, and b-GRE image acquisition was used to obtain 3D volume coverage of the patellofemoral and tibiofemoral cartilage and lower lumbar spine. Multiple T1-weighted images at various contrast times (spin-lock pulse duration [TSL]) were used to construct a T1 relaxation map in both phantoms and in the knee joint and spine in vivo. The transient signal decay during b-GRE image acquisition was corrected using a k-space filter. The T1-prepared b-GRE sequence was compared to a standard T1-prepared spin echo (SE) sequence and pulse sequence parameters were optimized numerically using the Bloch equations. Results:The b-GRE transient signal decay was found to depend on the initial T1-preparation and the corresponding T1 map was altered by variations in the point spread function with TSL. In a two compartment phantom, the steady state response was found to elevate T1 from 91.4 Ϯ 6.5 to 293.8 Ϯ 31 and 66.9 Ϯ 3.5 to 661 Ϯ 207 with no change in the goodness-of-fit parameter R 2 . Phase encoding along the longest cartilage dimension and a transient signal decay k-space filter retained T1 contrast. Measurement of T1 using the T1-prepared b-GRE sequence matches standard T1-prepared SE in the medial patellar and lateral patellar cartilage compartments. T1-preparedb-GRE T1 was found to have low interscan variability between four separate scans. Mean patellar cartilage T1 was elevated compared to femoral and tibial cartilage T1. Conclusion:The T1-prepared b-GRE acquisition rapidly and reliably accelerates T1 quantification of tissues offset partially by a TSL-dependent point spread function. CONVENTIONAL CONTRAST in magnetic resonance (MR) images derives from the 1 H magnetic relaxation properties of tissues. Variations in longitudinal magnetic relaxation (T1) and transverse magnetic relaxation (T2) distinguish the healthy and pathological states. An unconventional contrast mechanism based on the rotating frame spin-lattice relaxation time T1 (1) shows sensitivity to the diseased states of the human breast (2), early acute cerebral ischemia in rats (3,4), knee cartilage degeneration during osteoarthritis (5), posttraumatic cartilage injury (6), and narrowing of lumbar intervertebral discs associated with degenerative disc disease (7,8). In addition, functional T1 imaging shows both an augmented blood oxygen level dependent (BOLD) signal (9,10) and improved response to indirectly detected metabolic H 2 17 O (11-13). T1-weighted contrast is produced in vivo by allowing transverse magnetization to relax in the presence of an on-resonance continuous wave (cw) radio frequency (RF) pulse and is influenc...