Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers

Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones.

Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases

Analisis Kelayakan USAha Pembuatan Batu Bata dengan Tandan Kosong Kelapa Sawit sebagai Bahan Bakar di Desa Jentera Stabat Kecamatan Wampu Kabupaten Langkat

Tujuan penelitian adalah untuk mengetahui ketersediaan input (bahan baku, modal, tenaga kerja) USAha pembuatan batu bata dengan tandan kosong kelapa sawit sebagai bahan bakar, mengetahui besar pendapatannya, mengetahui tingkat kelayakan USAhanya, dan mengetahui dampak pemakaian tandan kosong kelapa sawit sebagai bahan bakar untuk USAha pembuatan batu bata di daerah penelitian. Penentuan daerah dilakukan secara purposive (sengaja) karena mempertimbangkan waktu dan jangkauan peneliti, sampel berjumlah 20 pengrajin batu bata yang ditentukan dengan metode sensus, data yang digunakan adalah data primer dan data sekunder. Metode penelitian yang digunakan untuk mengetahui ketersediaan input (bahan baku, modal, tenaga kerja) dan untuk mengetahui dampak pemakaian tandan kosong kelapa sawit sebagai bahan bakar yaitu menggunakan metode deskriptif, untuk mengetahui pendapatan menggunakan metode analisis pendapatan, untuk menganalisis kelayakan USAha menggunakan R/C Ratio dan BEP. Hasil penelitian menyimpulkan bahwa input (bahan baku, modal, tenaga kerja) cukup tersedia di daerah penelitian. Pendapatan USAha pembuatan batu bata adalah Rp 3.722.321,-/bulan atau Rp 644.277,-/10.000 batu bata. Diperoleh nilai R/C ratio > 1, BEP Produksi < Produksi, dan BEP Harga < Harga Jual. Pemakaian tandan kosong kelapa sawit sebagai bahan bakar dalam USAha pembuatan batu bata memberikan dampak positif, Dengan demikian dapat disimpulkan bahwa USAha pembuatan batu bata dengan tandan kosong kelapa sawit sebagai bahan bakar layak untuk diusahakan secara finansial di daerah penelitian

Evaluation of options for harvest of a recombinant E. coli fermentation producing a domain antibody using ultra scale-down techniques and pilot-scale verification

Ultra scale-down (USD) methods operating at the millilitre scale were used to characterise full-scale processing of E. coli fermentation broths autolysed to different extents for release of a domain antibody. The focus was on the primary clarification stages involving continuous centrifugation followed by depth filtration. The performance of this sequence was predicted by USD studies to decrease significantly with increased extents of cell lysis. The use of polyethyleneimine (PEI) reagent was studied to treat the lysed cell broth by precipitation of soluble contaminants such as DNA and flocculation of cell debris material. The USD studies were used to predict the impact of this treatment on the performance and here it was found that the fermentation could be run to maximum productivity using an acceptable clarification process (e.g a centrifugation stage operating at 0.11 L per m(2) equivalent gravity settling area per h followed by a resultant required depth filter area of 0.07 m(2) per L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. This article is protected by copyright. All rights reserved

Quantitative proteomics of rat livers shows that unrestricted feeding is stressful for proteostasis with implications on life span.

Studies in young mammals on the molecular effects of food restriction leading to prolong adult life are scares. Here, we used high-throughput quantitative proteomic analysis of whole rat livers to address the molecular basis for growth arrest and the apparent life-prolonging phenotype of the food restriction regimen. Over 1800 common proteins were significantly quantified in livers of ad libitum, restriction- and re-fed rats, which summed up into 92% of the total protein mass of the cells. Compared to restriction, ad libitum cells contained significantly less mitochondrial catabolic enzymes and more cytosolic and ER HSP90 and HSP70 chaperones, which are hallmarks of heat- and chemically-stressed tissues. Following re-feeding, levels of HSPs nearly reached ad libitum levels. The quantitative and qualitative protein values indicated that the restriction regimen was a least stressful condition that used minimal amounts of HSP-chaperones to maintain optimal protein homeostasis and sustain optimal life span. In contrast, the elevated levels of HSP-chaperones in ad libitum tissues were characteristic of a chronic stress, which in the long term could lead to early aging and shorter life span

Fed-Batch E. coli cultures in a shaken, single-use 24-well miniature bioreactor

At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes. Miniature bioreactors are becoming widely used in the biopharmaceutical industry as a tool for high throughput strain evaluation and fermentation process development. However, there are relatively few examples of miniature bioreactors capable of fed-batch operation and of supporting the high oxygen demand. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. In this work a shaken, single-use 24-well miniature bioreactor (Pall, Micro 24 MicroReactor System) has been characterised in terms of volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) to assess the feasibility of high cell density microbial cultures. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. To enable fed-batch cultivation in the miniature bioreactor system a bespoke feeding system for direct, continuous feed delivery has been developed that works at feed flow rates of 20μL h-1 and above. This feeding system allows for 24 fed-batch cultures to be run in parallel. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume; the oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency and gas flow rate over the range 3-101h-1. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.8-15.3s. It has been demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The bespoke feed delivery system was used to perform fed-batch cultures of an industrial E. coli strain producing an antibody fragment under operating conditions defined from the engineering characterisation studies. Fermentations were performed on a semi-complex medium containing glycerol with direct feeding of a glycerol solution initiated around 15 hours. It was found that direct feeding enhances biomass production by 30-40% and product expression by 45-65% in comparison to non-fed cultures. The feeding system developed in this work allows for industrially relevant microbial processes to be implemented at the microscale

Cardiac cell modelling: Observations from the heart of the cardiac physiome project

In this manuscript we review the state of cardiac cell modelling in the context of international initiatives such as the IUPS Physiome and Virtual Physiological Human Projects, which aim to integrate computational models across scales and physics. In particular we focus on the relationship between experimental data and model parameterisation across a range of model types and cellular physiological systems. Finally, in the context of parameter identification and model reuse within the Cardiac Physiome, we suggest some future priority areas for this field

Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70-Hsp40 Substrates.

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease

Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins.

During and after protein translation, molecular chaperones require ATP hydrolysis to favor the native folding of their substrates and, under stress, to avoid aggregation and revert misfolding. Why do some chaperones need ATP, and what are the consequences of the energy contributed by the ATPase cycle? Here, we used biochemical assays and physical modeling to show that the bacterial chaperones GroEL (Hsp60) and DnaK (Hsp70) both use part of the energy from ATP hydrolysis to restore the native state of their substrates, even under denaturing conditions in which the native state is thermodynamically unstable. Consistently with thermodynamics, upon exhaustion of ATP, the metastable native chaperone products spontaneously revert to their equilibrium non-native states. In the presence of ATPase chaperones, some proteins may thus behave as open ATP-driven, nonequilibrium systems whose fate is only partially determined by equilibrium thermodynamics