Publications by authors named "Sukyeong Lee"

33 Publications

Macrocyclic Immunoproteasome Inhibitors as a Potential Therapy for Alzheimer's Disease.

J Med Chem 2021 Aug 26;64(15):10934-10950. Epub 2021 Jul 26.

Department of Pharmaceutical Sciences, University of Kentucky, 789 South Limestone, Lexington, Kentucky 40536-0596, United States.

Previously, we reported that immunoproteasome (iP)-targeting linear peptide epoxyketones improve cognitive function in mouse models of Alzheimer's disease (AD) in a manner independent of amyloid β. However, these compounds' clinical prospect for AD is limited due to potential issues, such as poor brain penetration and metabolic instability. Here, we report the development of iP-selective macrocyclic peptide epoxyketones prepared by a ring-closing metathesis reaction between two terminal alkenes attached at the P2 and P3/P4 positions of linear counterparts. We show that a lead macrocyclic compound DB-60 () effectively inhibits the catalytic activity of iP in ABCB1-overexpressing cells (IC: 105 nM) and has metabolic stability superior to its linear counterpart. DB-60 () also lowered the serum levels of IL-1α and ameliorated cognitive deficits in Tg2576 mice. The results collectively suggest that macrocyclic peptide epoxyketones have improved CNS drug properties than their linear counterparts and offer promising potential as an AD drug candidate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.1c00291DOI Listing
August 2021

Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes.

Genome Med 2021 04 12;13(1):55. Epub 2021 Apr 12.

Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, 73104, USA.

Background: ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans.

Methods: To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants.

Results: We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles.

Conclusion: Our findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-021-00873-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042885PMC
April 2021

The Mitochondrial Protease LonP1 Promotes Proteasome Inhibitor Resistance in Multiple Myeloma.

Cancers (Basel) 2021 Feb 17;13(4). Epub 2021 Feb 17.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Multiple myeloma and its precursor plasma cell dyscrasias affect 3% of the elderly population in the US. Proteasome inhibitors are an essential part of several standard drug combinations used to treat this incurable cancer. These drugs interfere with the main pathway of protein degradation and lead to the accumulation of damaged proteins inside cells. Despite promising initial responses, multiple myeloma cells eventually become drug resistant in most patients. The biology behind relapsed/refractory multiple myeloma is complex and poorly understood. Several studies provide evidence that in addition to the proteasome, mitochondrial proteases can also contribute to protein quality control outside of mitochondria. We therefore hypothesized that mitochondrial proteases might counterbalance protein degradation in cancer cells treated with proteasome inhibitors. Using clinical and experimental data, we found that overexpression of the mitochondrial matrix protease LonP1 (Lon Peptidase 1) reduces the efficacy of proteasome inhibitors. Some proteasome inhibitors partially crossinhibit LonP1. However, we show that the resistance effect of LonP1 also occurs when using drugs that do not block this protease, suggesting that LonP1 can compensate for loss of proteasome activity. These results indicate that targeting both the proteasome and mitochondrial proteases such as LonP1 could be beneficial for treatment of multiple myeloma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13040843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922145PMC
February 2021

Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex.

Nat Commun 2021 01 12;12(1):281. Epub 2021 Jan 12.

Department of Biochemistry, University of Toronto, Toronto, ON, M5G 1M1, Canada.

A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20553-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804408PMC
January 2021

A biallelic pathogenic variant in the OGDH gene results in a neurological disorder with features of a mitochondrial disease.

J Inherit Metab Dis 2021 03 24;44(2):388-400. Epub 2020 Jun 24.

Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA.

2-Oxoglutarate dehydrogenase (OGDH) is a rate-limiting enzyme in the mitochondrial TCA cycle, encoded by the OGDH gene. α-Ketoglutarate dehydrogenase (OGDH) deficiency was previously reported in association with developmental delay, hypotonia, and movement disorders and metabolic decompensation, with no genetic data provided. Using whole exome sequencing, we identified two individuals carrying a homozygous missense variant c.959A>G (p.N320S) in the OGDH gene. These individuals presented with global developmental delay, elevated lactate, ataxia and seizure. Fibroblast analysis and modeling of the mutation in Drosophila were used to evaluate pathogenicity of the variant. Skin fibroblasts from subject # 2 showed a decrease in both OGDH protein and enzyme activity. Transfection of human OGDH cDNA in HEK293 cells carrying p.N320S also produced significantly lower protein levels compared to those with wild-type cDNA. Loss of Drosophila Ogdh (dOgdh) caused early developmental lethality, rescued by expressing wild-type dOgdh (dOgdh ) or human OGDH (OGDH ) cDNA. In contrast, expression to the mutant OGDH (OGDH ) or dOgdh carrying homologous mutations to human OGDH p.N320S variant (dOgdh ) failed to rescue lethality of dOgdh null mutants. Knockdown of dOgdh in the nervous system resulted in locomotion defects which were rescued by dOgdh expression but not by dOgdh expression. Collectively, the results indicate that c.959A>G variant in OGDH leads to an amino acid change (p.N320S) causing a severe loss of OGDH protein function. Our study establishes in the first time a genetic link between an OGDH gene mutation and OGDH deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jimd.12248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647956PMC
March 2021

LMP2 Inhibitors as a Potential Treatment for Alzheimer's Disease.

J Med Chem 2020 04 30;63(7):3763-3783. Epub 2020 Mar 30.

Department of Pharmaceutical Sciences, University of Kentucky, 789 South Limestone, Lexington, Kentucky 40536-0596, United States.

The immunoproteasome (iP), an inducible proteasome variant harboring three immunosubunits, low molecular mass polypeptide-2 (LMP2), multicatalytic endopeptidase complex subunit-1, and low molecular mass polypeptide-7 (LMP7), is involved in multiple facets of inflammatory responses. We recently reported that YU102, a dual inhibitor of the iP subunit LMP2 and the constitutive proteasome catalytic subunit β1, ameliorates cognitive impairments in mouse models of Alzheimer's disease (AD) independently of amyloid deposits. To investigate whether inhibition of LMP2 is sufficient to improve the cognitive functions of AD mice, here we prepared 37 YU102 analogues and identified a potent LMP2 inhibitor DB-310 () (IC: 80.6 nM) with improved selectivity and permeability in cells overexpressing ABCB1 transporters. We show that DB-310 induces suppression of IL-1α production in microglia cells and improves cognitive functions in the Tg2576 transgenic mouse model of AD. This study supports that inhibition of LMP2 is a promising therapeutic strategy for treatment of AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c00416DOI Listing
April 2020

Crystal Structure of the YcjX Stress Protein Reveals a Ras-Like GTP-Binding Protein.

J Mol Biol 2019 08 14;431(17):3179-3190. Epub 2019 Jun 14.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Stress proteins promote cell survival by monitoring protein homeostasis in cells and organelles. YcjX is a conserved protein of unknown function, which is highly upregulated in response to acute and chronic stress. Notably, heat shock induction of ycjX exceeded even levels observed for major stress-induced chaperones, including GroEL, ClpB, and HtpG, which use ATP as energy source. YcjX features a Walker-type nucleotide-binding domain indicating that YcjX might function as a molecular chaperone. Here, we present the first crystal structure of YcjX from Shewanella oneidensis solved at 1.9-Å resolution by SAD phasing. We show that YcjX is a GTP-binding protein that shares at its core the canonical alpha-beta domain of p21 (Ras). However, unlike Ras, YcjX features several unique insertions, including an entirely α-helical domain not previously observed in Ras-like GTPases. We note that this helical domain is reminiscent of a similar domain in the Gα subunit of heterotrimeric G proteins, supporting a potential role for YcjX as a signal transducer of stress responses. To elucidate the mechanism of GTP hydrolysis, we determined crystal structures of YcjX bound to GDP and GDPCP, respectively, which crystallized in three different nucleotide switch conformations. Supported by targeted mutagenesis experiments, we show that YcjX utilizes a non-canonical switch 2' motif not previously observed in Ras-like GTPases. Together, our structures provide atomic snapshots of YcjX in different functional states, illustrating the structural determinants for stress signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2019.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697224PMC
August 2019

Development of Novel Epoxyketone-Based Proteasome Inhibitors as a Strategy To Overcome Cancer Resistance to Carfilzomib and Bortezomib.

J Med Chem 2019 05 19;62(9):4444-4455. Epub 2019 Apr 19.

Department of Pharmaceutical Sciences , University of Kentucky , Lexington , Kentucky 40536 , United States.

Over the past 15 years, proteasome inhibitors (PIs), namely bortezomib, carfilzomib (Cfz) and ixazomib, have significantly improved the overall survival and quality-of-life for multiple myeloma (MM) patients. However, a significant portion of MM patients do not respond to PI therapies. Drug resistance is present either de novo or acquired after prolonged therapy through mechanisms that remain poorly defined. The lack of a clear understanding of clinical PI resistance has hampered the development of next-generation PI drugs to treat MM patients who no longer respond to currently available therapies. Here, we designed and synthesized novel epoxyketone-based PIs by structural modifications at the P1' site. We show that a Cfz analog, 9, harboring a hydroxyl substituent at its P1' position was highly cytotoxic against cancer cell lines displaying de novo or acquired resistance to Cfz. These results suggest that peptide epoxyketones incorporating P1'-targeting moieties may have the potential to bypass resistance mechanisms associated with Cfz and to provide additional clinical options for patients resistant to Cfz.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b01943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675178PMC
May 2019

Cryo-EM Structures of the Hsp104 Protein Disaggregase Captured in the ATP Conformation.

Cell Rep 2019 01;26(1):29-36.e3

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Hsp104 is a ring-forming, ATP-driven molecular machine that recovers functional protein from both stress-denatured and amyloid-forming aggregates. Although Hsp104 shares a common architecture with Clp/Hsp100 protein unfoldases, different and seemingly conflicting 3D structures have been reported. Examining the structure of Hsp104 poses considerable challenges because Hsp104 readily hydrolyzes ATP, whereas ATP analogs can be slowly turned over and are often contaminated with other nucleotide species. Here, we present the single-particle electron cryo-microscopy (cryo-EM) structures of a catalytically inactive Hsp104 variant (Hsp104) in the ATP-bound state determined between 7.7 Å and 9.3 Å resolution. Surprisingly, we observe that the Hsp104 hexamer adopts distinct ring conformations (closed, extended, and open) despite being in the same nucleotide state. The latter underscores the structural plasticity of Hsp104 in solution, with different conformations stabilized by nucleotide binding. Our findings suggest that, in addition to ATP hydrolysis-driven conformational changes, Hsp104 uses stochastic motions to translocate unfolded polypeptides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.12.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347426PMC
January 2019

Structural insights into the oligomerization of FtsH periplasmic domain from Thermotoga maritima.

Biochem Biophys Res Commun 2018 01 24;495(1):1201-1207. Epub 2017 Nov 24.

School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea; Steitz Center for Structural Biology, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea. Electronic address:

Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. The transmembrane protein, FtsH, is the only known ATP-dependent protease responsible for this task. The mechanisms by which FtsH recognizes, unfolds, translocates, and proteolyzes its substrates remain unclear. The structure and function of the ATPase and protease domains of FtsH have been previously characterized while the role of the FtsH periplasmic domain has not clearly identified. Here, we report the 1.5-1.95 Å resolution crystal structures of the Thermotoga maritima FtsH periplasmic domain (tmPD) and describe the dynamic features of tmPD oligomerization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2017.11.158DOI Listing
January 2018

Structural determinants for protein unfolding and translocation by the Hsp104 protein disaggregase.

Biosci Rep 2017 12 22;37(6). Epub 2017 Dec 22.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, U.S.A.

The ring-forming Hsp104 ATPase cooperates with Hsp70 and Hsp40 molecular chaperones to rescue stress-damaged proteins from both amorphous and amyloid-forming aggregates. The ability to do so relies upon pore loops present in the first ATP-binding domain (AAA-1; loop-1 and loop-2 ) and in the second ATP-binding domain (AAA-2; loop-3) of Hsp104, which face the protein translocating channel and couple ATP-driven changes in pore loop conformation to substrate translocation. A hallmark of loop-1 and loop-3 is an invariable and mutational sensitive aromatic amino acid (Tyr and Tyr) involved in substrate binding. However, the role of conserved aliphatic residues (Lys, Lys, and Val) flanking the pore loop tyrosines, and the function of loop-2 in protein disaggregation has not been investigated. Here we present the crystal structure of an N-terminal fragment of Hsp104 exhibiting molecular interactions involving both AAA-1 pore loops, which resemble contacts with bound substrate. Corroborated by biochemical experiments and functional studies in yeast, we show that aliphatic residues flanking Tyr and Tyr are equally important for substrate interaction, and abolish Hsp104 function when mutated to glycine. Unexpectedly, we find that loop-2 is sensitive to aspartate substitutions that impair Hsp104 function and abolish protein disaggregation when loop-2 is replaced by four aspartate residues. Our observations suggest that Hsp104 pore loops have non-overlapping functions in protein disaggregation and together coordinate substrate binding, unfolding, and translocation through the Hsp104 hexamer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BSR20171399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741831PMC
December 2017

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation.

Sci Rep 2017 09 11;7(1):11184. Epub 2017 Sep 11.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA.

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conflicting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We find that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specific substrates, such as yeast prions, which likely depends on a direct N domain interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-11474-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593927PMC
September 2017

Short-Term Effect of Convenience Meal Intake on Glycemic Response and Satiety among Healthy College Students in South Korea.

Clin Nutr Res 2017 Jul 18;6(3):215-220. Epub 2017 Jul 18.

Department of Food and Nutrition, Korea National University of Transportation, Jeungpyeong 27909, Korea.

This study examined the effect of convenience meals purchased at convenience stores on glycemic response and satiety in healthy college students. A total of 9 non-obese volunteers (4 males and 5 females) aged 20 to 24 years participated in this study. On 3 separate days, participants consumed a standard diet (cooked rice and side dishes), type 1 convenience meal (kimbap and instant ramen), and type 2 convenience meal (sweet bread and flavored milk). Capillary blood-glucose response and satiety were measured every 30 minutes for 2 hours after consuming the 3 different test meals. Although mean fasting glucose levels were not different, glucose levels at 30 minutes and 120 minutes after the type 1 convenience meal intake were significantly higher than those in the standard meal (p < 0.05, p < 0.01). Total glucose response was higher after consumption of the type 1 convenience meal, followed by the type 2 convenience meal and standard meal (p < 0.05). Though the type 2 convenience meal contained higher calorie than the other meals, satiety of the type 2 convenience meal was lowest at 30 minutes and 60 minutes after consumption (p < 0.01, p < 0.05). This pilot study suggests that convenience meals may increase glycemic response or induce higher calorie intake with low satiety compared with nutritionally balanced Korean style meal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7762/cnr.2017.6.3.215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539215PMC
July 2017

Structural Elements Regulating AAA+ Protein Quality Control Machines.

Front Mol Biosci 2017 4;4:27. Epub 2017 May 4.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of MedicineHouston, TX, USA.

Members of the ATPases Associated with various cellular Activities (AAA+) superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS) motif, and the Pre-Sensor I insert (PS-I) motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmolb.2017.00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415569PMC
May 2017

2.4 Å resolution crystal structure of human TRAP1NM, the Hsp90 paralog in the mitochondrial matrix.

Acta Crystallogr D Struct Biol 2016 08 13;72(Pt 8):904-11. Epub 2016 Jul 13.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

TRAP1 is an organelle-specific Hsp90 paralog that is essential for neoplastic growth. As a member of the Hsp90 family, TRAP1 is presumed to be a general chaperone facilitating the late-stage folding of Hsp90 client proteins in the mitochondrial matrix. Interestingly, TRAP1 cannot replace cytosolic Hsp90 in protein folding, and none of the known Hsp90 co-chaperones are found in mitochondria. Thus, the three-dimensional structure of TRAP1 must feature regulatory elements that are essential to the ATPase activity and chaperone function of TRAP1. Here, the crystal structure of a human TRAP1NM dimer is presented, featuring an intact N-domain and M-domain structure, bound to adenosine 5'-β,γ-imidotriphosphate (ADPNP). The crystal structure together with epitope-mapping results shows that the TRAP1 M-domain loop 1 contacts the neighboring subunit and forms a previously unobserved third dimer interface that mediates the specific interaction with mitochondrial Hsp70.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798316009906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973209PMC
August 2016

Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate.

Proc Natl Acad Sci U S A 2016 Mar 29;113(11):2952-7. Epub 2016 Feb 29.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1516167113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801263PMC
March 2016

Molecular chaperones: guardians of the proteome in normal and disease states.

F1000Res 2015 15;4. Epub 2015 Dec 15.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.

Proteins must adopt a defined three-dimensional structure in order to gain functional activity, or must they? An ever-increasing number of intrinsically disordered proteins and amyloid-forming polypeptides challenge this dogma. While molecular chaperones and proteases are traditionally associated with protein quality control inside the cell, it is now apparent that molecular chaperones not only promote protein folding in the "forward" direction by facilitating folding and preventing misfolding and aggregation, but also facilitate protein unfolding and even disaggregation resulting in the recovery of functional protein from aggregates. Here, we review our current understanding of ATP-dependent molecular chaperones that harness the energy of ATP binding and hydrolysis to fuel their chaperone functions. An emerging theme is that most of these chaperones do not work alone, but instead function together with other chaperone systems to maintain the proteome. Hence, molecular chaperones are the major component of the proteostasis network that guards and protects the proteome from damage. Furthermore, while a decline of this network is detrimental to cell and organismal health, a controlled perturbation of the proteostasis network may offer new therapeutic avenues against human diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/f1000research.7214.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754035PMC
February 2016

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Proc Natl Acad Sci U S A 2013 May 6;110(21):8513-8. Epub 2013 May 6.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the species-specific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1217988110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666692PMC
May 2013

Structural basis for intersubunit signaling in a protein disaggregating machine.

Proc Natl Acad Sci U S A 2012 Jul 16;109(31):12515-20. Epub 2012 Jul 16.

Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.

ClpB is a ring-forming, ATP-dependent protein disaggregase that cooperates with the cognate Hsp70 system to recover functional protein from aggregates. How ClpB harnesses the energy of ATP binding and hydrolysis to facilitate the mechanical unfolding of previously aggregated, stress-damaged proteins remains unclear. Here, we present crystal structures of the ClpB D2 domain in the nucleotide-bound and -free states, and the fitted cryoEM structure of the D2 hexamer ring, which provide a structural understanding of the ATP power stroke that drives protein translocation through the ClpB hexamer. We demonstrate that the conformation of the substrate-translocating pore loop is coupled to the nucleotide state of the cis subunit, which is transmitted to the neighboring subunit via a conserved but structurally distinct intersubunit-signaling pathway common to diverse AAA+ machines. Furthermore, we found that an engineered, disulfide cross-linked ClpB hexamer is fully functional biochemically, suggesting that ClpB deoligomerization is not required for protein disaggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1207040109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411974PMC
July 2012

Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine.

J Struct Biol 2012 Aug 24;179(2):172-80. Epub 2012 May 24.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Hsp104 is a double ring-forming AAA+ ATPase, which harnesses the energy of ATP binding and hydrolysis to rescue proteins from a previously aggregated state. Like other AAA+ machines, Hsp104 features conserved cis- and trans-acting elements, which are hallmarks of AAA+ members and are essential to Hsp104 function. Despite these similarities, it was recently proposed that Hsp104 is an atypical AAA+ ATPase, which markedly differs in 3D structure from other AAA+ machines. Consequently, it was proposed that arginines found in the non-conserved M-domain, but not the predicted Arg-fingers, serve the role of the critical trans-acting element in Hsp104. While the structural discrepancy has been resolved, the role of the Arg-finger residues in Hsp104 remains controversial. Here, we exploited the ability of Hsp104 variants featuring mutations in one ring to retain ATPase and chaperone activities, to elucidate the functional role of the predicted Arg-finger residues. We found that the evolutionarily conserved Arg-fingers are absolutely essential for ATP hydrolysis but are dispensable for hexamer assembly in Hsp104. On the other hand, M-domain arginines are not strictly required for ATP hydrolysis and affect the ATPase and chaperone activities in a complex manner. Our results confirm that Hsp104 is not an atypical AAA+ ATPase, and uses conserved structural elements common to diverse AAA+ machines to drive the mechanical unfolding of aggregated proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2012.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411918PMC
August 2012

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

J Biol Chem 2011 Feb 8;286(6):4404-11. Epub 2010 Dec 8.

Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.158741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039362PMC
February 2011

CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation.

Proc Natl Acad Sci U S A 2010 May 19;107(18):8135-40. Epub 2010 Apr 19.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. Whereas the ability to disaggregate proteins is dependent on the Hsp104 M-domain, the location of the M-domain is controversial and its exact function remains unknown. Here we present cryoEM structures of two Hsp104 variants in both crosslinked and noncrosslinked form, in addition to the structure of a functional Hsp104 chimera harboring T4 lysozyme within the M-domain helix L2. Unexpectedly, we found that our Hsp104 chimera has gained function and can solubilize heat-aggregated beta-galactosidase (beta-gal) in the absence of the Hsp70 system. Our fitted structures confirm that the subunit arrangement of Hsp104 is similar to other AAA+ machines, and place the M-domains on the Hsp104 exterior, where they can potentially interact with large, aggregated proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1003572107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889573PMC
May 2010

An intersubunit signaling network coordinates ATP hydrolysis by m-AAA proteases.

Mol Cell 2009 Sep;35(5):574-85

Institute for Genetics, Center for Molecular Medicine Cologne, University of Cologne, Germany.

Ring-shaped AAA+ ATPases control a variety of cellular processes by substrate unfolding and remodeling of macromolecular structures. However, how ATP hydrolysis within AAA+ rings is regulated and coupled to mechanical work is poorly understood. Here we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes, conserved AAA+ machines in the inner membrane of mitochondria. ATP binding to one AAA subunit inhibits ATP hydrolysis by the neighboring subunit, leading to coordinated rather than stochastic ATP hydrolysis within the AAA ring. Unbiased genetic screens define an intersubunit signaling pathway involving conserved AAA motifs and reveal an intimate coupling of ATPase activities to central AAA pore loops. Coordinated ATP hydrolysis between adjacent subunits is required for membrane dislocation of substrates, but not for substrate processing. These findings provide insight into how AAA+ proteins convert energy derived from ATP hydrolysis into mechanical work.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2009.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744646PMC
September 2009

Crystallization and preliminary X-ray crystallographic analysis of a 40 kDa N-terminal fragment of the yeast prion-remodeling factor Hsp104.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 Sep 31;63(Pt 9):784-6. Epub 2007 Aug 31.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 A. Native 2 diffracted to 2.9 A resolution and belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 A. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1744309107038328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2376311PMC
September 2007

Visualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB.

Mol Cell 2007 Jan;25(2):261-71

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2007.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855157PMC
January 2007

M domains couple the ClpB threading motor with the DnaK chaperone activity.

Mol Cell 2007 Jan;25(2):247-60

ZMBH, Universität Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany.

The AAA(+) chaperone ClpB mediates the reactivation of aggregated proteins in cooperation with the DnaK chaperone system. ClpB consists of two AAA domains that drive the ATP-dependent threading of substrates through a central translocation channel. Its unique middle (M) domain forms a coiled-coil structure that laterally protrudes from the ClpB ring and is essential for aggregate solubilization. Here, we demonstrate that the conserved helix 3 of the M domain is specifically required for the DnaK-dependent shuffling of aggregated proteins, but not of soluble denatured substrates, to the pore entrance of the ClpB translocation channel. Helix 3 exhibits nucleotide-driven conformational changes possibly involving a transition between folded and unfolded states. This molecular switch controls the ClpB ATPase cycle by contacting the first ATPase domain and establishes the M domain as a regulatory device that acts in the disaggregation process by coupling the threading motor of ClpB with the DnaK chaperone activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2006.11.008DOI Listing
January 2007

The E3 ubiquitin ligase CHIP binds the androgen receptor in a phosphorylation-dependent manner.

Biochim Biophys Acta 2006 Jun 4;1764(6):1073-9. Epub 2006 Apr 4.

Program in Structural and Computational Biology and Molecular Biophysics, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030, USA.

In Eukarya, the 26S proteasome is primarily responsible for intracellular protein degradation. To be degraded, proteins must be ubiquitinated. The latter requires a multi-enzyme cascade consisting of an E1, an E2, and an E3 enzyme. While there is only a single E1 and a few E2s, there are many different E3s that target substrates by recognizing specific sequence motifs, known as degrons. Here, we have used the peptide array technology to identify binding motifs in the human androgen receptor (AR), which are recognized by the Carboxyl-terminus of Hsc70-Interacting Protein (CHIP), a U-box E3 and Hsp70/Hsp90 co-chaperone. We show that CHIP recognizes AR in a highly specific, phosphorylation- and sequence-dependent manner, and propose that this interaction could provide a mechanism that regulates the degradation of CHIP substrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2006.03.013DOI Listing
June 2006

Molecular chaperones in protein quality control.

J Biochem Mol Biol 2005 May;38(3):259-65

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Conversely, protein aggregation and misfolding are primary contributors to many devastating human diseases, such as prion-mediated infections, Alzheimer's disease, type II diabetes and cystic fibrosis. While the native conformation of a polypeptide is encoded within its primary amino acid sequence and is sufficient for protein folding in vitro, the situation in vivo is more complex. Inside the cell, proteins are synthesized or folded continuously; a process that is greatly assisted by molecular chaperones. Molecular chaperones are a group of structurally diverse and mechanistically distinct proteins that either promote folding or prevent the aggregation of other proteins. With our increasing understanding of the proteome, it is becoming clear that the number of proteins that can be classified as molecular chaperones is increasing steadily. Many of these proteins have novel but essential cellular functions that differ from that of more "conventional" chaperones, such as Hsp70 and the GroE system. This review focuses on the emerging role of molecular chaperones in protein quality control, i.e. the mechanism that rids the cell of misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5483/bmbrep.2005.38.3.259DOI Listing
May 2005

Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB.

Cell 2004 Nov;119(5):653-65

Zentrum für Molekulare Biologie der Universität Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany.

Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2004.11.027DOI Listing
November 2004
-->