Complete structure of an epithelial keratin dimer: implications for intermediate filament assembly

Keratins are cytoskeletal proteins that hierarchically arrange into filaments, starting with the dimer sub-unit. They are integral to the structural support of cells, in skin, hair and nails. In skin, keratin is thought to play a critical role in conferring the barrier properties and elasticity of skin. In general, the keratin dimer is broadly described by a tri-domain structure: a head, a central rod and a tail. As yet, no atomistic-scale picture of the entire dimer structure exists; this information is pivotal for establishing molecular-level connections between structure and function in intermediate filament proteins. The roles of the head and tail domains in facilitating keratin filament assembly and function remain as open questions. To address these, we report results of molecular dynamics simulations of the entire epithelial human K1/K10 keratin dimer. Our findings comprise: (1) the first three-dimensional structural models of the complete dimer unit, comprising of the head, rod and tail domains; (2) new insights into the chirality of the rod-domain twist gained from analysis of the full domain structure; (3) evidence for tri-subdomain partitioning in the head and tail domains; and, (4) identification of the residue characteristics that mediate non-covalent contact between the chains in the dimer. Our findings are immediately applicable to other epithelial keratins, such as K8/K18 and K5/K14, and to intermediate filament proteins in general

Structural Dynamics of the Vimentin Coiled-Coil Contact Regions involved in Filament Assembly as revealed by Hydrogen-Deuterium Exchange

Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They not only are responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil−forming proteins exhibiting a characteristic central α-helical domain (″rod″). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also, a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and investigate the dynamic properties of the full-length proteins in solution during the various assembly steps, we analyzed the patterns of hydrogen-deuterium exchange (HDex) in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of theα-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within full-length vimentin, in particular in regions of contact in α-helical and linker segments that stabilize different oligomeric forms such as tetramers, ULFs, and mature filaments. Moreover, HDex analysis of the point-mutated variants directly demonstrated the active role of the IF-consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately, using molecular dynamics simulation procedures, we provide a structural model for the subdomain-mediated tetramer−tetramer interaction via ″cross-coiling″ as the first step of the assembly process

Computational and theoretical modeling of intermediate filament networks: Structure, mechanics and disease

Intermediate filaments, in addition to microtubules and actin microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells. It was discovered during the recent decades that in most cells, intermediate filament proteins play key roles to reinforce cells subjected to large-deformation, and that they participate in signal transduction, and it was proposed that their nanomechanical properties are critical to perform those functions. However, it is still poorly understood how the nanoscopic structure, as well as the combination of chemical composition, molecular structure and interfacial properties of these protein molecules contribute to the biomechanical properties of filaments and filament networks. Here we review recent progress in computational and theoretical studies of the intermediate filaments network at various levels in the protein’s structure. A multiple scale method is discussed, used to couple molecular modeling with atomistic detail to larger-scale material properties of the networked material. It is shown that a finer-trains-coarser methodology as discussed here provides a useful tool in understanding the biomechanical property and disease mechanism of intermediate filaments, coupling experiment and simulation. It further allows us to improve the understanding of associated disease mechanisms and lays the foundation for engineering the mechanical properties of biomaterials.United States. Air Force Office of Scientific ResearchNational Science Foundation (U.S.)United States. Office of Naval Researc

Role of intermediate filament desmin in development of desmin-related myopathy

Desmin is a major intermediate filament of muscle cells, serving to transmit mechanical forces and propagate mechanochemical signals, to coordinate contraction and relaxation cycles, and to stabilize the positioning of cellular organelles, e.g. mitochondria. Around 70 desmin gene mutations have been reported in conjunction with desmin-related myopathy. Desmin-related myopathy can be described as pathophysiological complex, accompanied by desmin intracellular aggregate accumulation and impairment of desmin interactions with structural proteins, signal molecules, and cell organelles. However, the precise molecular mechanism underlying desmin-related myopathy have not been described yet. There are speculations if it is connected with toxic effects of desmin aggregates or with violation of desmin mechanotransduction functions. The general aim of the present PhD project was to extend existing knowledge about the molecular machinery on how desmin gene mutations lead to the development of desmin-related myopathy, with an emphasis on development of cardiomyopathies. To address this aim the following research questions were stated: (i) genetic study of a group of patients with cardiomyopathies in order to describe novel mutations in the desmin gene, and to assess the frequency of DES A213V; (ii) genetic study by means of next-generation sequencing approach of a group of patients with idiopathic restrictive cardiomyopathy in order to describe novel genetic variants associated with disease; (iii) functional study of desmin gene point mutations effect on mitochondrial properties. The main findings regarding genetic background were: (i) DES A213V represents a disease-modifying polymorphism, rather than disease-related mutation, since it was found both in patients and healthy donors; (ii) combination of disease-related– disease-modifying or disease-related–disease-related genetic variants, rather than single disease-related mutation, determined the development of idiopathic restrictive cardiomyopathy. Most proteins of these combinations belonged to four functional groups: sarcomeric contractile proteins, mechanosensing Z-disc proteins, nuclear membrane, and outer mitochondrial membrane proteins. Functional studies of the impact of desmin mutations on mitochondria showed that aggregate-prone mutations decreased mitochondrial calcium uptake, as well as depressed maximal oxygen consumption rate and spare respiratory capacity. In contrast, non-aggregate-prone mutations did not disturb mitochondrial calcium. They did, however, result in the reduction of maximal oxygen consumption rate and affected spare respiratory capacity. To conclude, (i) distortion of desmin mechanotransduction functions plays an important role in desmin-related myopathy onset, affecting mitochondrial properties; (ii) combination of mutations in genes encoding sarcomeric contractile and mechanosensing proteins, rather than a single mutation, predisposes to the development of cardiomyopathy. These data facilitate understanding of molecular pathways underlying desmin-related myopathy development, and increase existing knowledge of intracellular interactions within the muscle cell

From lamins to lamina: a structural perspective

Lamin proteins are the major constituents of the nuclear lamina, a proteinaceous network that lines the inner nuclear membrane. Primarily, the nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes, including DNA replication and repair, regulation of gene expression, and signaling. Mutations in human lamin A and associated proteins were found to cause a large number of diseases, termed ‘laminopathies.' These diseases include muscular dystrophies, lipodystrophies, neuropathies, and premature aging syndromes. Despite the growing number of studies on lamins and their associated proteins, the molecular organization of lamins in health and disease is still elusive. Likewise, there is no comprehensive view how mutations in lamins result in a plethora of diseases, selectively affecting different tissues. Here, we discuss some of the structural aspects of lamins and the nuclear lamina organization, in light of recent result

RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo

Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins over-expressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of a-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.Peer reviewe

Structural determinants of intermediate filament mechanics

Intermediate filaments (IFs) are integral to the cell cytoskeleton, supporting cellular mechanical stability. Unlike other cytoskeletal components, the detailed structure of assembled IFs has yet to be resolved. This review highlights new insights, linking the complex IF hierarchical assembly to their mechanical properties and impact on cellular functions. While we focus on vimentin IFs, we draw comparisons to keratins, showcasing the distinctive structural and mechanical features that underlie their unique mechanical responses

라민 조립의 분자 메커니즘과 노화 방지를 위한 기능성 식품의 개발

학위논문(박사)--서울대학교 대학원 :농업생명과학대학 농생명공학부,2020. 2. 하남출.Intermediate filament (IFs) proteins form flexible fibrous structures that provide vital mechanical support in cells of higher eukaryotes. Nuclear structure and function are governed by lamins, which are intermediate filaments mostly consisting of α-helices. Therefore, it is necessary to determine the mechanism of lamin assembly to understand the dynamic functions of the nuclear membrane. Different lamin assembly models have been proposed based on low resolution or fragmented structures. However, their assembly mechanisms at the molecular level are poorly understood. Here, I present a crystal structure of a long human lamin fragment at a 3.2 Å resolution, which allows for the visualization of the full-length features. The structure shows the anti-parallel arrangement of two coiled-coil dimers, which is important for the assembly process. The anti-parallel interaction corresponded to the A11 interaction of vimentin. Furthermore, another new interaction (eA22) involved in longitudinal elongation of lamin filament was discovered by using chemical cross-linking and mass analysis. Based on these two interactions (A11 and eA22 interaction), a molecular mechanism of lamin assembly was proposed that is in agreement with a recent model representing the native state and could explain pathological mutations. Furthermore, it was observed that the altered binding strength of eA22 interaction when the mutations were introduced to the residues at a, d position of coil 1a to discover the role of coil 1a on the longitudinal extension of filament. The result indicated that the binding affinity of eA22 interactions is altered depending on the stability of coil 1a dimer. Consistent results were obtained in the nuclear shapes and the distribution of the lamin A/C when the mutant lamin genes were overexpressed in the cell lines. This study provides an insight to understand the role of coil 1a as a molecular switch in the eA22 interaction, which contributes to the molecular basis for the development of a new treatment against laminopathies. A silent mutation of the lamin A/C gene, leading to the production of a C-terminally truncated protein (called progerin), causes Hutchinson-Gilford progeria syndrome (HGPS). A deformed nuclear shape is a hallmark of HGPS cells, which shares with normal aged cells. A recent study found that progerin makes a direct interaction with a middle fragment of lamin A/C, which leads to nuclear deformation. A synthetic compound (called JH4), blocking the interaction between the progerin and meddle fragment of lamin A/C, ameliorated the aging phenotypes both in cells and mouse models. The natural compounds were discovered that interrupt progerin-mediated interaction. Of these, the flavonoid morin was investigated further characterization. Morin showed an effect on the interruption of the progerin-mediated interaction. Treatment of morin to progerin-expressing cells and HGPS cells alleviated the nuclear deformation, which is comparable to the previous synthetic compound JH4. Since morin is a naturally occurring substance with the antioxidant activity, it has the potential to develop as a functional food to improve lifespan and health in both HGPS patients and normal aged persons.중간 필라멘트(Intermediate Filament) 단백질은 고등진핵생물 세포에 필수적이며, 외부에서 가해지는 힘에 대해 물리적인 지지를 제공하는 유연한 섬유 구조를 형성한다. 핵의 구조와 기능은 중간 필라멘트에 속하는 라민에 의해 조절된다. 따라서 핵의 구조와 기능에 대해 이해하기 위해서는 라민의 조립 메커니즘 규명이 필수적이다. 하지만 짧은 조각의 라민 구조만 규명되어 왔으며, 전자현미경을 이용한 낮은 해상도를 가지는 구조의 한계에 의해 라민의 조립 메커니즘에 대한 분자적 수준의 이해가 부족한 상황이었다. 본 연구에서는 라민의 전체 길이를 보여줄 만큼 긴 결정 구조를 3.2 Å 해상도로 규명하였다. 결정 구조에서는 두개의 코일형 이중체 (coiled-coil dimers)가 나란한 방향으로 배열되어 사중체를 형성하고 있는 것을 확인할 수 있었는데, 이는 기존 중간 필라멘트 비멘틴에서 관찰된 A11 결합과 유사하였다. 더 나아가 케미컬 크로스링커와 질량 분석법을 적용하여 라민 필라멘트의 연장에 관여하는 새로운 결합 (eA22 interaction)을 발견하였다. 이러한 두가지 결합 (A11, eA22 결합)을 통해 라민 조립 메커니즘을 분자적으로 제시하였으며, 이는 최근 제시된 초저온 전자토모그래피 (Electron cryotomography; Cryo-ET) 이미지와도 일치하였다. 또한 필라멘트가 세로방향으로 확장될 때 코일 1a의 역할을 밝히기 위해 코일 1a의 a와 d 위치에 존재하는 아미노산 잔기의 변형을 도입하여 eA22 결합의 결합강도가 변화되는 것을 관찰하였다. 실험 결과 코일 1a의 안정도에 따라서 eA22 결합이 변화된다는 것을 확인할 수 있었다. 여러 세포에 돌연변이 라민 유전자를 과발현시켜 핵의 모양과 라민 단백질의 분포를 관찰하였을 때에도 일관된 결과를 얻었다. 이러한 연구는 코일 1a가 eA22 결합을 위한 분자적 스위치로 역할을 한다는 것을 시사하며 라민과 관련된 질병을 위한 치료제 개발에 분자적 기반을 마련할 것으로 기대한다. 라민 A/C 유전자의 무성 돌연변이 (silent mutation) 가 발생하면 C-말단이 잘려진 상태의 단백질인 프로제린 (progerin)이 생성되며 이는 허친슨-길포드 프로게리아 증후군 (Hutchinson-Gilford progeria syndrome; HGPS)을 일으킨다. 기형적인 핵 모양은 HGPS 세포의 특징으로, 정상적인 노화 세포에서도 이와 같은 특징이 나타난다. 최근 연구에서는 프로제린이 라민 A/C의 중간 파편과 직접 상호작용하여 핵변형을 일으킨다는 사실이 밝혀졌다. 해당 상호작용을 차단하는 합성 화합물(JH4)은 세포와 마우스 모델 모두에서 노화 표현형을 개선하는 효과를 보였다. 본 연구에서는 프로제린과 정상적인 라민의 결합을 억제하는 천연물을 스크리닝하였다. 여러가지 후보물질 중 플라보노이드 계열의 모린을 최종 후보물질로 선택하였다. 모린은 프로제린과 라민의 결합을 억제하는 효과를 나타내었다. 프로제린을 과발현시킨 세포와 HGPS 세포에 모린을 처리하였더니 핵이 변형되는 현상이 완화되었고, 이는 기존에 합성된 JH4 물질과 비슷한 수준으로 효과를 보였다. 모린은 항산화 효과를 가지고 있는 자연물질이기 때문에 모린을 기능성 식품으로 개발한다면 HGPS 환자들뿐만 아니라 정상적인 노화 작용에 대하여 수명과 건강을 향상시키는 효과를 기대할 수 있을 것이다.Chapter 1. Introduction 14 1.1. Intermediate filament 14 1.1.1. The domain organization of intermediate filament 14 1.1.2. Classification of intermediate filament proteins 15 1.2. Nuclear lamins 16 1.2.1. Expression of nuclear lamins 16 1.2.2. The domain organization of lamins 17 1.2.3. Post-translational modification of lamins 17 1.2.4. Assembly of lamins 19 1.3. Functions of lamins 21 1.3.1. Protein interactions 21 1.3.2. Chromatin organization 22 1.3.3. DNA damage and repair 24 1.3.4. Cellular signaling 24 1.3.5. Transcription 25 1.4. Laminopathies 27 1.4.1. Emery Dreifuss muscular dystrophy (EDMD) 27 1.4.2. Dilated Cardiomyopathy (DCM) and Limb Girdle Muscular Dystrophy type 1B (LGMD1B) 28 1.4.3. Dunnigan-type familial partial lipodystrophy (FPLD) 29 1.4.4. Hutchinson-Gilford progeria syndrome (HPGS) 30 1.5. Purpose of Research 31 Chapter 2. Structural basis for lamin assembly at the molecular level 33 2.1. Introduction 34 2.2. Materials and Methods 38 2.2.1. Plasmid construction 38 2.2.2. Purification of the recombinant proteins 40 2.2.3. Crystallization and structure determination 41 2.2.4. SEC-MALS 45 2.2.5. Pull-down assays 45 2.2.6. Cross-linking reaction and MS/MS analysis 46 2.2.7. Isothermal Titration Calorimetry 53 2.2.8. Immunofluorescence staining 53 2.3. Results 55 2.3.1. Structural determination of the lamin fragment 55 2.3.2. Antiparallel interaction between two coiled coils 66 2.3.3. The lamin A11 interaction for the lamin assembly process 73 2.3.4. Enhanced binding of coil 2 by the A11 interaction 77 2.3.5. The eA22 interaction 81 2.3.6. Proposed lamin assembly model at the molecular level 90 2.3.7. Laminopathies correlated with A11 and eA22 interactions 97 2.4. Discussion 111 Chapter 3. The separation of lamin coil 1a for the eA22 interaction and its implications to laminopathies 113 3.1. Introduction 114 3.2. Materials and Methods 117 3.2.1. Plasmid construction 117 3.2.2. Purification of the recombinant proteins 118 3.2.3. Pull-down assays 119 3.2.4. Isothermal Titration Calorimetry 120 3.2.5. Immunofluorescence staining 121 3.3. Results 122 3.3.1. The highly conserved sequence of coil 1a 122 3.3.2. The two possible conformational change of coil 1a 126 3.3.3. The role of hydrophobic residues of coil 1a on the eA 22 interaction 130 3.3.4. Structure and biochemical analysis of the role of coil 1a on eA22 interaction 136 3.3.5. The correlation between laminopathies and eA22 interaction 139 3.4. Discussion 142 Chapter 4. The flavonoid morin alleviates nuclear deformation of the aged cells by interrupting the progerin-lamin A/C binding 145 4.1. Introduction 146 4.2. Materials and Methods 148 4.2.1. Plasmids construction and purification 148 4.2.2. Purification of the recombinant proteins 148 4.2.3. Modified ELISA assay 149 4.2.4. Isothermal Titration Calorimetry 150 4.2.5. Immunoprecipitation and Western-blot analysis 151 4.3. Results 152 4.3.1. Screening of inhibitors of progerin-lamin A binding in the natural compound library 152 4.3.2. The morin inhibits the interaction between Ig-like domain of lamin A and progerin 155 4.3.3. The flavonoid morin ameliorates the nuclear deformation of progerin-expressing cells 160 4.3.4. Morin ameliorates the nuclear deformation of HGPS cells 166 4.4. Discussion 173 Bibliography 175 Abstract in Korean 204Docto

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To determine whether a tag would affect de novo filament assembly, we used vimentin fused at the N-terminus with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) to assemble into filaments in two vimentin-deficient epithelial cells, MCF-7 and A431. We showed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with β-catenin at cell–cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin at a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by only untagged vimentin. The data suggest that placing a tag at the N-terminus may create steric hinderance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Taken together our results shows that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into a pre-existing network